PGAM1 Inhibitors and Methods Related Thereto

ABSTRACT

In certain embodiments, the disclosure relates to methods of treating or preventing a PGAM1 mediated condition such as cancer or tumor growth comprising administering an effective amount of PGAM1 inhibitor, for example, an anthracene-9,10-dione derivative to a subject in need thereof. In certain embodiments, the disclosure relates to methods of treating or preventing cancer, such as lung cancer, head and neck cancer, and leukemia, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a compound disclosed herein or pharmaceutically acceptable salt to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/847,670filed Dec. 19, 2017, which is a division of U.S. application Ser. No.14/349,550 filed Apr. 3, 2014, which is the National Stage ofInternational Application No. PCT/US2012/059740 filed Oct. 12, 2012,which claims the benefit of U.S. Provisional Application No. 61/547,278filed Oct. 14, 2011. The entirety of each of these applications ishereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA120272 andCA140515 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 11147USDIV2_ST25.txt. The text file is 1 KB, wascreated on Sep. 19, 2019, and is being submitted electronically viaEFS-Web.

BACKGROUND

There remains a need for improved therapeutics useful in the treatmentof cancer. The Warburg effect in cancer cells consists of an increase inaerobic glycolysis and enhanced lactate production, which generates moreATPs more quickly than in normal cells that overwhelmingly rely onoxidative phosphorylation. In addition, tumor tissue traps more glucosethan normal tissue does, as cancer cells use elevated amounts of glucoseas a carbon source for anabolic biosynthesis of macromolecules. Theseinclude nucleotides, amino acids and fatty acids, to produce RNA/DNA,proteins and lipids, respectively, which are used for cell proliferationand to fulfill the request of the rapidly growing tumors. Interestingly,leukemia cells are also highly glycolytic, despite that such cellsreside within the bloodstream at higher oxygen tensions than cells inmost normal tissues, as well as tumor cells that commonly reside inhypoxia. This suggests that tumor hypoxia may not be a major contributorto select for cells dependent on anaerobic metabolism.

During glycolysis, glycolytic intermediates includingglucose-6-phosphate (G6P) can be diverted into the pentose phosphatepathway (PPP), which contributes to macromolecular biosynthesis byproducing reducing potential in the form of reduced nicotinamide adeninedinucleotide phosphate (NADPH) and/or ribose-5-phosphate (R5P), thebuilding blocks for nucleotide synthesis. NADPH is the most crucialmetabolite produced by the PPP because NADPH not only fuelsmacromolecular biosynthesis such as lipogenesis, but it also functionsas a crucial antioxidant, quenching the reactive oxygen species (ROS)produced during rapid proliferation of cancer cells.

Glycolysis and glutaminolysis supply the carbon input required for theTCA cycle to function as a biosynthetic ‘hub’ and permits the productionof other macromolecules including amino acids and fatty acids. Thus,cancer cells appear to coordinate glycolysis and anabolism to provide anoverall metabolic advantage to cancer cell proliferation and diseasedevelopment.

Engel et al., report that a phosphoglycerate mutase-derived polypeptideinhibits glycolytic flux and induces cell growth arrest in tumor celllines. J Biol Chem, 2004, 279, 35803-35812.

Evans et al., report the mechanistic and structural requirements foractive site labeling of phosphoglycerate mutase by spiroepoxides. SeeMol. BioSyst., 2007, 3, 495-506.

PCT Patent Application PCT/US2009/000257, published as WO 2010/082912 A1discloses certain disulfonamide derivatives. The disclosure alsodiscloses methods for treating tumors and cancer.

SUMMARY

In certain embodiments, the disclosure relates to methods of treating orpreventing a PGAM1 mediated condition such as cancer or tumor growthcomprising administering an effective amount of PGAM1 inhibitor, forexample, an anthracene-9,10-dione derivative to a subject in needthereof.

In certain embodiments, the anthracene-9,10-dione derivative is acompound of Formula I,

prodrug, ester, or salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each individually andindependently hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl,carbocyclyl, aryl, or heterocyclyl, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ are optionally substituted with one or more, the same ordifferent, R⁹;

R⁹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁹ is optionally substituted with one or more, thesame or different, R¹¹;

R¹⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁹ is optionally substituted with one or more, thesame or different, R¹¹;

R¹¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R⁷ is hydroxyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R⁸ is hydroxyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino wherein R¹ is optionally substitutedwith one or more, the same or different, R⁹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring is optionally substituted with one or more, thesame or different R¹⁰.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with an alkyl,wherein the alkyl group is optionally substituted with one or more, thesame or different R¹¹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with a methyl,wherein the methyl group is optionally substituted with one or more, thesame or different R¹¹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with a methyl,wherein the methyl group is substituted with one or more, the same ordifferent halogens.

In some embodiments the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position withtrifluoromethane.

In certain embodiments, the derivative is3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid or3,4-dihydroxy-9,10-dioxo-N-(4-(trifluoromethyl)phenyl)-9,10-dihydroanthracene-2-sulfonamideprodrug, ester, or salt thereof optionally substituted with one or more,the same or different, substituent(s).

In certain embodiments, the compound comprises a Log P of greater than2, 3, or 4.

In some embodiments, the disclosure relates to pharmaceuticalcompositions of compounds of Formula I or salts thereof.

In some embodiments, the disclosure relates to pharmaceuticalcompositions of compounds of Formula I containing a pharmaceuticallyacceptable excipient or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to pharmaceuticalcompositions of compounds of Formula I containing a pharmaceuticallyacceptable excipient or a pharmaceutically acceptable salt thereof and asecond therapeutic agent.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition of Formula I to a subject diagnosed with, exhibitingsymptoms of, or at risk of cancer.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition of Formula I to a subject diagnosed with, exhibitingsymptoms of, or at risk of cancer wherein the pharmaceuticalcompositions is administered in combination with a secondchemotherapeutic agent.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition of Formula I to a subject diagnosed with, exhibitingsymptoms of, or at risk of cancer in combination with a secondanti-cancer agent such as gefitinib, erlotinib, docetaxel, cis-platin,5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin,daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin andmithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol,taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin,bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide,cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole,letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab,cetuximab, dasatinib, imatinib, bevacizumab, combretastatin,thalidomide, and/or lenalidomide or combinations thereof.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition of Formula I to a subject diagnosed with, exhibitingsymptoms of, or at risk of cancer wherein the cancer is selected fromthe group consisting of leukemia, cervical, ovarian, colon, breast,gastric, lung, skin, ovarian, pancreatic, prostate, head, neck, andrenal cancer.

In some embodiments, the disclosure relates to the use of a compound ofFormula I in the production of a medicament for the treatment orprevention of cancer.

In certain embodiments, the disclosure relates to an antibody that bindsthe PGAM1 phospho-Y26 epitope. In certain embodiments the antibody is ahuman chimera or a humanized antibody that binds PGAM1 phospho-Y26epitope. In certain embodiments, the disclosure contemplates the use ofa pharmaceutical composition comprising an antibody that binds PGAM1phospho-Y26 epitope in the treatment of cancer in combination with otheranti-cancer agent by administering an effective amount to a subject inneed thereof.

In certain embodiments, the disclosure also relates to the method ofsynthesis of compounds disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows data indicating PGAM1 controls intracellular 3-PG levelsin cancer cells and is important for glycolysis and anabolicbiosynthesis, as well as cell proliferation and tumor growth.Intracellular concentrations of 3-PG were determined in diverse PGAM1knockdown cancer cells and compared to control cells.

FIG. 1B shows data for 2-PG.

FIG. 1C shows H1299 cells with stable knockdown of PGAM1 and controlcells harboring an empty vector were tested for glycolytic rate.

FIG. 1D shows lactate production.

FIG. 1E shows RNA biosynthesis.

FIG. 1F shows lipogenesis.

FIG. 1G shows NADPH/NADP+ ratio.

FIG. 1H shows oxidative PPP flux.

FIG. 1I shows the intracellular ATP levels in the presence or absence of100 nM oligomycin (ATP synthase inhibitor) were also tested.

FIG. 1J shows oxygen consumption rate (J).

FIG. 1K shows cell proliferation rates determined by cell counting indiverse human cancer (H1299, 212LN and MDA-MB231) and leukemia (KG1a,Molm14 and K562) cells with stable knockdown of PGAM1, which werenormalized to the corresponding control cells harboring an empty vector.

FIG. 1L shows Stable knockdown of PGAM1 by shRNA attenuates tumor growthpotential of H1299 cells in xenograft nude mice. Left: Dissected tumors(indicated by red arrows) in a representative nude mouse and expressionof PGAM1 in tumor lysates are shown. Right: PGAM1 knockdown cells showsignificantly reduced tumor formation in xenograft nude mice compared tocells harboring empty vector control.

FIG. 2A shows data indicating attenuation of PGAM1 results in increasedintracellular levels of 3-PG, which binds to and inhibits 6PGD bycompeting with its substrate 6-PG. Enzyme activity of 6PGD in H1299 celllysates was determined in the presence of increasing concentrations of3-PG. Relative 6PGD activity was normalized to the control sampleswithout 3-PG treatment. 3-PG levels in control H1299 cells with emptyvector and PGAM1 knockdown are 62.5±10.8 μM and 256±41.9 μM,respectively.

FIG. 2B shows recombinant 6PGD (r6PGD).

FIG. 2C show thermal shift melting curves of 6PGD and 3PG. Thermal shiftassay was performed to examine the protein (6PGD) and “ligand” (3PG)interaction. Change of melting temperature (Tm) in a dose-dependentmanner at concentrations from 100 μM to 25 mM demonstrates that 3-PGdirectly binds to the protein. Kd for 6PGD-3-PG interaction wasdetermined to be 460±40 μM.

FIG. 2D show the Dixon plot indicating that 3-PG inhibits 6PGD and thedissociation constant (Ki) was determined.

FIG. 2E shows the Lineweaver-Burk plot shows that 3-PG functions as acompetitive inhibitor of 6PGD.

FIG. 2F shows thermal shift melting curves of 6PGD and 6PG. Thermalshift assay was performed to examine the protein (6PGD) and ligand (6PG)interaction. Change of melting temperature (Tm) in a dose-dependentmanner at concentrations from 5 μM to 5 mM demonstrates that 6-PGdirectly binds to the protein. Kd for 6PGD-6PG interaction wasdetermined to be 37±3 μM.

FIG. 3 illustrates a proposed model: role of PGAM1 in cancer cellmetabolism. Top: PGAM1 activity is upregulated in cancer cells topromote glycolysis and keep the intracellular 3-PG levels low, which inturn permits high levels of the PPP and biosynthesis to fulfill therequest of rapidly growing tumors. PGAM1 also maintains thephysiological levels of 2-PG to sustain PHGDH activity, which diverts3-PG from glycolysis to serine synthesis and contributes to maintainingrelatively low levels of 3-PG in cancer cells. These effects in concertprovide a metabolic advantage to cancer cell proliferation and tumorgrowth. Bottom: When PGAM1 is inhibited, 3-PG levels are elevated, whichin turn inhibit 6PGD and consequently the oxidative PPP and anabolicbiosynthesis. At the same time, 2-PG is decreased to levels below thephysiological concentrations, leading to decreased PHGDH activity, whichfacilitates 3-PG accumulation. Such metabolic changes result inattenuated cell proliferation and tumor development.

FIG. 4A shows data indicating rescue of reduced 2-PG levels in PGAM1knockdown cells reverses the phenotypes due to attenuation of PGAM1.2-PG levels in diverse cancer cells with stable knockdown of PGAM1 weredetermined in the presence and absence of cell permeable methyl-2-PG.

FIG. 4B shows H1299 cells with stable knockdown of PGAM1 were tested forlactate production in the presence and absence of methyl-2-PG.

FIG. 4C shows oxidative PPP flux.

FIG. 4D shows biosynthesis of RNA.

FIG. 4E shows lipids.

FIG. 4F shows cell proliferation.

FIG. 5A shows data indicating rescue of reduced 2-PG levels due to PGAM1attenuation results in decreased 3-PG levels by activating PHGDH. 3-PGlevels in diverse cancer cells with stable knockdown of PGAM1 weredetermined in the presence and absence of methyl-2-PG.

FIG. 5B shows enzyme activity of PHGDH in PGAM1 knockdown H1299 (left)or 212LN (right) cell lysates determined in the presence of increasingconcentrations of 2-PG. Relative enzyme activity was normalized to thecontrol samples without 2-PG treatment. 2-PG levels in control H1299cells with empty vector and PGAM1 knockdown cells are 46.2±10.2 μM and15.0±14.1 μM, respectively, while 2-PG levels in 212LN cells with emptyvector and stable knockdown of PGAM1 are 58.3±20.1 μM and 17.8±14.4 μM,respectively.

FIG. 5C shows recombinant PHGDH (rPHGDH).

FIG. 5D shows serine biosynthesis rate of H1299 cells with stableknockdown of PGAM1 was determined by measuring 14C incorporation intoserine from 14C-glucose in the presence and absence of methyl-2-PG.Relative serine biosynthesis was normalized to control cells harboringan empty vector without methyl-2-PG treatment.

FIG. 5E shows western blot result indicating shRNA-mediated knockdown ofPHGDH in H1299 cells with stable knockdown of PGAM1 in the presence orabsence of methyl-2-PG treatment.

FIG. 5F shows 2-PG (left) and 3-PG (right) levels in PGAM1 knockdowncells upon PHGDH knockdown were determined in the presence and absenceof methyl-2-PG.

FIG. 5G shows PGAM1 stable knockdown cells treated with or without shRNAtargeting PHGDH were tested for PPP flux (G) in the presence and absenceof methyl-2-PG.

FIG. 5H shows biosynthesis of serine, lipids and RNA (left, middle andright, respectively).

FIG. 6A shows illustrations and data on identification andcharacterization of small molecule PGAM1 inhibitor, PGMI-004A. Schematicrepresentation of the primary and secondary screening strategies toidentify lead compounds as PGAM1 inhibitors.

FIG. 6B shows structure of alizarin and its derivatives alizarin Red Sand PGAM inhibitor (PGMI)-004A.

FIG. 6C shows PGMI-004A inhibits PGAM1 with an IC50 of 13.1 μM, whichwas determined by incubating purified human PGAM1 proteins withincreasing concentrations of PGMI-004A. The error bars represent meanvalues+/−SD from three replicates of each sample.

FIG. 6D shows Kd value was determined as 7.2±0.7 μM by incubatingpurified human PGAM1 proteins with increasing concentrations ofPGMI-004A. The fluorescence intensity (Ex: 280 nm, em: 350 nm) fromTryptophan was measured.

FIG. 6E shows competitive binding assay of PGMI-004A with recombinantPGAM1 protein in the presence of increasing concentrations of PGAM1substrate 3-PG. Increased free PGAM1 was determined by an increase influorescence intensity.

FIG. 6F shows Dixon plot analysis of PGAM1 enzyme assay in the presenceof different concentrations of PGMI-004A and 3-PG. The reaction velocity(v) was determined by the rate of the decrease in fluorescence (ex: 340nm, em: 460 nm) by NADH oxidation. Ki was determined to be 3.91±2.50 μM.

FIG. 6G shows thermal shift melting curves of PGAM1 and PGMI-004A.Thermal shift assay was performed to examine the protein (PGAM1) and“ligand” (inhibitor PGMI-004A) interaction. Change of meltingtemperature (Tm) in a dose-dependent manner at concentrations from 2.5μM to 80 μM demonstrates that PGMI-004A directly binds to the protein.Kd for PGAM1-PGMI-004A interaction was determined to be 9.4±2.0 μM.

FIG. 7A shows data indicating inhibition of PGAM1 by PGMI-004A revealsthat PGAM1 enzyme activity is important for regulation of 3-PG and 2-PGlevels and coordination of glycolysis and biosynthesis to promote cancercell proliferation. 2-PG (left) and 3-PG (right) levels in H1299 cellstreated with or without PGMI-004A were determined in the presence andabsence of methyl-2-PG.

FIG. 7B shows lactate production in H1299 cells treated with or withoutPGMI-004A were determined in the presence and absence of methyl-2-PG.

FIG. 7C shows intracellular ATP levels.

FIG. 7D shows H1299 cells treated with or without PGMI-004A were testedfor oxidative PPP flux in the presence and absence of methyl-2-PG.

FIG. 7E shows NADPH/NADP+ ratio.

FIG. 7F shows H1299 cells treated with or without PGMI-004A were testedfor biosynthesis of lipids.

FIG. 7G show RNA.

FIG. 7H shows cell proliferation.

FIG. 7I shows cell viability of H1299 cells in the presence ofincreasing concentrations of PGMI-004A. Cell viability was determined bytrypan blue exclusion.

FIG. 7J shows diverse human leukemia cells.

FIG. 7K shows and control human dermal fibroblasts (HDF) cells.

FIG. 8A shows data indicating that PGMI-004A treatment results inincreased 3-PG and decreased 2-PG levels, and reduced cell proliferationof primary leukemia cells from human patients, as well as attenuatedtumor growth in xenograft nude mice in vivo. Tumor growth in xenograftnude mice injected with H1299 cells were compared between the group ofmice treated with PGMI-004A and the control group treated with vehiclecontrol. p values were determined by a two-tailed Student's t test.

FIG. 8B shows tumor size.

FIG. 8C shows PGAM1 protein expression (lower) and enzyme activity(upper) levels were examined using primary leukemia cells from diversehuman patients with AML, CIVIL and B-ALL and compared to controlperipheral blood cells from healthy donors.

FIG. 8D shows effect of PGMI-004A treatment on 3-PG (left) and 2-PG(right) levels in human primary leukemia cells isolated from peripheralblood samples from a representative AML patient.

FIG. 8E show effect of PGMI-004A treatment on cell viability (left),PGAM1 activity (middle) and lactate production (right) in human primaryleukemia cells from a representative CML patient.

FIG. 8F shows effect of methyl-2-PG treatment on decreased cellviability (left) in PGMI-004A-treated human primary leukemia cells fromAML patient.

FIG. 8G shows lactate production.

FIG. 8H shows PGMI-004A indicating no toxicity in treatment (120h) ofperipheral blood cells from representative healthy human donors.

FIG. 8I shows CD34+ cells isolated from bone marrow samples.

FIG. 9A illustrates and show data on alizarin and alizarin Red S asPAGM1 inhibitors. Screen identifies alizarin and its derivative alizarinRed S as PGAM1 inhibitors. Primary screen was performed as an in vitroPGAM1 assay using recombinant PGAM1 identified 5 lead compounds aspotential PGAM1 inhibitors.

FIG. 9B shows secondary screen was performed as an in vitro enolaseassay using recombinant enolase to exclude potential off target effectsof the lead compounds.

FIG. 9C shows three compounds were identified as PGAM1 inhibitorsincluding hexachlorophene, p-hydroxycinnamaldehyde and alizarin.Inhibitory potency of different lead compounds including Alizarin,hexachlorophene and p-hydroxycinnamaldehyde in human leukemia KG1acells. Cells were treated with individual compounds for 4h.

FIG. 9D shows cell viability of KG1a cells in the presence of increasingconcentrations of Alizarin (72h). Cell viability was determined bytrypan blue exclusion.

FIG. 9E shows chemical structures of commercially available alizarinderivatives.

FIG. 9F shows relative PGAM enzyme activity.

FIG. 10A illustrates certain embodiments and show data on PAGM1inhibitors. (Chemical structures of specially designed derivatives ofalizarin Red S, including PGMI-001A to 5A.

FIG. 10B shows inhibitory effects of diverse alizarin derivatives onenzyme activity of recombinant PGAM1 in an in vitro PGAM1 enzyme assay.

FIG. 10C shows PGMI-004A demonstrates more potent activity in regard toPGAM1 inhibition in KG1a cells compared to controls including alizarin,alizarin Red S and PGMI-001A (2h).

FIG. 10D shows rescue of 2-PG levels in PGMI-004A-treated H1299 cells bytreatment with methyl-2-PG results in increased lactate productioncompared with control cells treated with PGMI-004A, while this rescuedphenotype was abolished when enolase was knocked down or inhibited byspecific inhibitor NaF.

FIG. 10E shows effect of PGMI-004A treatment on cell proliferation oflung cancer H1299.

FIG. 10F shows leukemia KG1a.

FIG. 10G shows head and neck cancer 212LN cells.

FIG. 10H shows diverse human leukemia cells. Cells were treated withincreasing concentrations of PGMI-004A for 72h.

FIG. 10I shows PGMI-004A treatment does not affect cell proliferation ofhuman foreskin fibroblasts (HFF), human HaCaT keratinocyte cells andhuman melanocyte PIG1 cells.

FIG. 11A show data indicating rescue of reduced 2-PG levels bymethyl-2PG treatment results in decreased 3-PG levels and rescuesdecreased biosynthesis and cell proliferation in PGAM1 knockdown breastcancer cells. Intracellular ATP levels in control and PGAM1 knockdownH1299 cells in the presence and absence of methyl-2-PG.

FIG. 11B shows effect of treatment with methyl-2-PG on PPP flux inMDA-MB231 cells with stable knockdown of PGAM1 compared to controlcells.

FIG. 11C shows biosynthesis of RNA.

FIG. 11D shows lipids.

FIG. 11E shows cell proliferation.

FIG. 12A shows data indicating PGMI-004A effectively inhibits tumorgrowth in xenograft nude mice and cell viability of primary leukemiacells from human patients. Histological morphology of hematoxylin-eosinstained tissue sections of representative nude mice in PGMI-004A orvehicle control-treated groups (#39 and #46, respectively). Nude micewere treated daily with PGMI-004A (100 mg/kg/day) intraperitoneally for7 days. Peripheral blood samples were collected and applied for analysisof hematological properties. The vital organs were collected forhisto-pathological analysis. Histopathologic tissue sections (kidney)from representative nude mice stained with hematoxylin-eosin did notreveal significant differences between the vehicle and PGMI-004A treatedgroups. Images were analyzed and captured using ImageScope software(Aperio Technologies Inc.) without any additional or subsequent imageprocessing (high power images are 20×; low power images are either 4.0×,4.2×, or 4.4×). Scale bars are indicated.

FIG. 12B shows lung.

FIG. 12C shows liver.

FIG. 12D shows spleen.

FIG. 12E shows dissected tumors (indicated by arrows) in representativenude mice treated with vehicle control or PGMI-004A are shown.

FIG. 12F Tumors from two groups of xenograft nude mice treated witheither vehicle control or PGMI-004A are shown.

FIG. 13A shows data indicating Y26 phosphorylation of PGAM1 is common inleukemia cells, which contributes to control of 3-PG and 2-PG levels andis important for cancer cell metabolism, proliferation and tumor growth.PGAM1 is commonly expressed and Y26-phosphorylated in leukemia andmultiple myeloma cells.

FIG. 13B shows left: Active, recombinant FGFR1 (rFGFR1) and JAK2 (rJAK2)phosphorylates rPGAM1 at Y26 and such phosphorylation is abolished inY26F mutant proteins. Right: Inhibition of FOP2-FGFR1 by TKI258 inleukemia KG1a cells.

FIG. 13C shows JAK2 V617F mutant by AG490 in leukemia HEL cells resultsin decreased Y26 phosphorylation of PGAM1.

FIG. 13D shows generation of H1299 cells with stable knockdown ofendogenous hPGAM1 and rescue expression of mPGAM1 WT or Y26F mutant.

FIG. 13E shows attenuation of PGAM1 by expressing catalytically lessactive mPGAM1 Y26F mutant results in increased intracellular 3-PG levels(left) and decreased 2-PG levels (right), while treatment with cellpermeable methyl-2PG reverses these alterations in Y26F cells.

FIG. 13F shows Y26F cells have a decreased cell proliferation ratecompared to control cells expressing mPGAM1 WT, while treatment withmethyl-2PG significantly rescues the reduced cell proliferation of Y26Fcells.

FIG. 13G shows attenuation of PGAM1 by rescue expression of Y26F mutantresults in decreased tumor growth potential of H1299 cells in xenograftnude mice. Left: Dissected tumors (indicated by red arrows) in arepresentative nude mouse; expression and Y26 phosphorylation levels ofPGAM1 or mPGAM1 proteins in tumor lysates are shown. Right: Cellsexpressing mPGAM1 Y26F show significantly reduced tumor formation inxenograft nude mice compared to cells expressing mPGAM1 WT.

DETAILED DESCRIPTION

The Warburg effect in cancer cells consists of increased aerobicglycolysis and enhanced lactate production, which generates more ATPsmore quickly than in normal cells that overwhelmingly rely on oxidativephosphorylation. In addition, cancer cells use glycolytic intermediatesfor anabolic biosynthesis of macromolecules. These include nucleotides,amino acids and fatty acids, to produce RNA/DNA, proteins and lipids,respectively, which are necessary for cell proliferation and to fulfillthe request of the rapidly growing tumors.

PGAM1 converts 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG)during glycolysis. This is a unique step in glycolysis as most of theglycolytic intermediates that are used as precursors for anabolicbiosynthesis are upstream of this step. In many cancers, includinghepatocellular carcinoma and colorectal cancer, PGAM1 activity isincreased compared to that in the normal tissues. PGAM1 gene expressionis believed to be upregulated due to loss of TP53 in cancer cells, asTP53 negatively regulates PGAM1 gene expression.

Inhibition of PGAM1 results in increased 3-PG and decreased 2-PG levelsin cancer cells, leading to significantly decreased PPP flux andbiosynthesis, and consequently reduced cell proliferation and tumorgrowth. Y26 phosphorylation of PGAM1 is common in human leukemias.Leukemogenic tyrosine kinases (LTKs) are constitutively activated andfrequently implicated in pathogenesis of human leukemias, includingFGFR1 fusions associated 8p11 stem cell MPD, BCR-ABL associated CIVIL,FLT3-ITD associated AML and JAK2 V617F associated myeloproliferativedisorders. Y26 phosphorylation activates PGAM1 by promoting His 11phosphorylation and contributes to control of 3-PG and 2-PG levels,providing a novel, acute mechanism underlying PGAM1 upregulation inaddition to chronic changes regulated by TP53. Shutting off or forcedactivation of glycolytic enzymes may disrupt not only energy productionbut also supplies of metabolic intermediates as precursors foranabolism, both of which are required for cancer cells to survive, growand proliferate.

PGAM1 protein expression, Y26 phosphorylation and enzyme activity levelsare upregulated in human primary leukemia cells compared with normalperipheral blood cells from healthy donors. Disclosed herein are certainPGAM1 inhibitors which effectively inhibit cancer/leukemia cellproliferation, tumor growth in xenograft nude mice.

Terms

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to10 carbon atoms, while the term “lower alkyl” or “C₁₋₄alkyl” has thesame meaning as alkyl but contains from 1 to 4 carbon atoms. The term“higher alkyl” has the same meaning as alkyl but contains from 7 to 20carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl,n-nonyl, and the like; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively). Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the like; while unsaturated carbocycles include cyclopentenyl andcyclohexenyl, and the like.

“Heterocarbocycles” or heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur which may be saturated or unsaturated (but notaromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized. Heterocarbocycles include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Aryl” means an aromatic carbocyclic monocyclic or polycyclic ring suchas phenyl or naphthyl. Polycyclic ring systems may, but are not requiredto, contain one or more non-aromatic rings, as long as one of the ringsis aromatic.

As used herein, “heteroaryl” refers an aromatic heterocarbocycle having1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, andcontaining at least 1 carbon atom, including both mono- and polycyclicring systems. Polycyclic ring systems may, but are not required to,contain one or more non-aromatic rings, as long as one of the rings isaromatic. Representative heteroaryls are furyl, benzofuranyl,thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl,pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl,pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that theuse of the term “heteroaryl” includes N-alkylated derivatives such as a1-methylimidazol-5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkylthio” refers to an alkyl group as defined above attached through asulfur bridge. An example of an alkylthio is methylthio, (i.e., —S—CH₃).

“Alkoxy” refers to an alkyl group as defined above attached through anoxygen bridge. Examples of alkoxy include, but are not limited to,methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy,n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy,n-propoxy, propoxy, n-butoxy, s-butoxy, t-butoxy.

“Alkylamino” refers an alkyl group as defined above attached through anamino bridge. An example of an alkylamino is methylamino, (i.e.,—NH—CH₃).

“Alkanoyl” refers to an alkyl as defined above attached through acarbonyl bride (i.e., —(C═O)alkyl).

“Alkylsulfonyl” refers to an alkyl as defined above attached through asulfonyl bridge (i.e., —S(═O)₂alkyl) such as mesyl and the like, and“Arylsulfonyl” refers to an aryl attached through a sulfonyl bridge(i.e., —S(═O)₂aryl).

“Alkylsulfinyl” refers to an alkyl as defined above attached through asulfinyl bridge (i.e. —S(═O)alkyl).

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NR_(a)R_(b), —NR_(a)C(═O)R_(b),—NR_(a)C(═O)NR_(a)NR_(b), —NR_(a)C(═O)OR_(b), —NR_(a)SO₂R_(b),—C(═O)R_(a), —C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OC(═O)NR_(a)R_(b),—OR_(a), —SR_(a), —SOR_(a), —S(═O)₂R_(a), —OS(═O)₂R_(a) and—S(═O)₂OR_(a). R_(a) and R_(b) in this context may be the same ordifferent and independently hydrogen, halogen hydroxyl, alkyl, alkoxy,alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

As used herein, “salts” refer to derivatives of the disclosed compoundswhere the parent compound is modified making acid or base salts thereof.Examples of salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkylamines, ordialkylamines; alkali or organic salts of acidic residues such ascarboxylic acids; and the like. In preferred embodiment the salts areconventional nontoxic pharmaceutically acceptable salts including thequaternary ammonium salts of the parent compound formed, and non-toxicinorganic or organic acids. Preferred salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

“Subject” refers any animal, preferably a human patient, livestock,rodent, monkey or domestic pet.

The term “prodrug” refers to an agent that is converted into abiologically active form in vivo. Prodrugs are often useful because, insome situations, they may be easier to administer than the parentcompound. They may, for instance, be bioavailable by oral administrationwhereas the parent compound is not. The prodrug may also have improvedsolubility in pharmaceutical compositions over the parent drug. Aprodrug may be converted into the parent drug by various mechanisms,including enzymatic processes and metabolic hydrolysis.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur or nitrogen atom or replacing an amino group with ahydroxyl group or vice versa. The derivative may be a prodrug.Derivatives may be prepare by any variety of synthetic methods orappropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) MichaelB. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F.Tietze hereby incorporated by reference.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g., patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” can be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It can alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

The half maximal inhibitory concentration (IC50) refers to a measure ofthe effectiveness of a compound in inhibiting biological or biochemicalfunction. This quantitative measure indicates how much of a particulardrug or other substance (inhibitor) is needed to inhibit a givenbiological process (or component of a process, i.e. an enzyme, cell,cell receptor or microorganism) by half.

The partition coefficient is a ratio of concentrations of un-ionizedcompound between the two solutions. To measure the partition coefficientof ionizable solutes, the pH of the aqueous phase is adjusted such thatthe predominant form of the compound is un-ionized. The logarithm of theratio of the concentrations of the un-ionized solute in the solvents(octane/water) refers to the log P. The log P value is a measure oflipophilicity.

Phosphoglycerate Mutase 1 (PGAM1) PGAM1 Coordinates Glycolysis andBiosynthesis to Promote Tumor Growth

Cancer cells coordinate glycolysis and biosynthesis to support rapidlygrowing tumors Experiments herein indicate that glycolytic enzymephosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancersdue to loss of TP53, contributes to biosynthesis regulation in part bycontrolling intracellular levels of its substrate 3-phosphoglycerate(3-PG) and product 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits6-phosphogluconate dehydrogenase in the oxidative pentose phosphatepathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase toprovide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA orsmall molecule inhibitors, such as PGMI-004A, results in increased 3-PGand decreased 2-PG levels in cancer cells, leading to significantlydecreased glycolysis, PPP flux and biosynthesis, as well as attenuatedcell proliferation and tumor growth.

Experiments herein indicate that upregulation of PGAM1 by increased geneexpression in cancer cells provides a metabolic advantage to cancer cellproliferation and tumor growth; PGAM1 coordinates glycolysis andanabolic biosynthesis, at least in part by controlling intracellularlevels of its substrate 3-PG and product 2-PG (FIG. 3). Although it isnot intended that embodiments of the disclosure be limited by anyparticular mechanism, it is believed that 3-PG inhibits 6PGD by directlybinding to the active site of 6PGD and competing with its substrate6-PG. Attenuation of PGAM1 results in abnormal accumulation of 3-PG,which in turn inhibits 6PGD and consequently the oxidative PPP andanabolic biosynthesis. PGAM1 controls the intracellular levels of itsproduct 3-PG not only directly through substrate consumption but alsoindirectly by controlling levels of its product 2-PG. Physiologicalconcentrations of 2-PG promote the enzyme activity of PHGDH, whichconverts 3-PG to pPYR, reducing the cellular 3-PG levels. Uponattenuation of PGAM1, 2-PG is decreased to levels below thephysiological concentrations, leading to decreased PHGDH activity, whichfacilitates 3-PG accumulation. This represents a regulatory mechanism bywhich 2-PG activates PHGDH to provide feedback control of 3-PG levels.Thus, PGAM1 activity is upregulated in cancer cells to promoteglycolysis and keep the intracellular 3-PG levels low, which in turnpermits high levels of the PPP and biosynthesis to fulfill the requestof rapidly growing tumors. This is consistent with a report thatexpression of TP53 suppresses oxidative PPP in cancer cells (Jiang etal., Nature cell biology, 2011, 13, 310-316). In addition, PGAM1 mayalso be responsible for maintaining the physiological levels of 2-PG tosustain PHGDH activity, which diverts 3-PG from glycolysis to serinesynthesis and contributes to maintaining relatively low levels of 3-PGin cancer cells.

Inhibition of PGAM1 by shRNA or treatment with a small moleculeinhibitor PGMI-004A results in altered glycolysis and anabolicbiosynthesis, and reduced cancer cell proliferation and tumor growth.Interestingly, targeting PGAM1 does not significantly affectintracellular ATP levels. Decreased ATP production due to attenuatedglycolysis in PGAM1 knockdown cells may be compensated by alternativemechanisms other than mitochondrial oxidative phosphorylation, orperhaps the ATP consumption in PGAM1 knockdown cells is decreasedaccordingly. Methyl-2-PG treatment rescues most of the aforementionedphenotypes. Rescued 2-PG levels in cells with attenuated PGAM1 reverseddecreased lactate production by rescuing the glycolytic processdownstream of PGAM1, as well as reduced oxidative PPP flux andbiosynthesis of RNA and lipids, at least in part by decreasing elevated3-PG levels. However, methyl-2-PG treatment only partially rescues theattenuated cell proliferation in PGAM1 knockdown cells or cells treatedwith PGMI-004A. This result suggests that PGAM1 may contribute to cellproliferation in both 2-PG-dependent and independent manners.

The current understanding of the connection between glycolysis andPPP/biosynthesis is based upon a model in which glycolytic intermediatescan be diverted into PPP and biosynthesis pathways as precursors. Theconcentrations of glycolytic metabolites such as 3-PG and 2-PG candirectly affect the catalytic activity of enzymes involved in PPP andbiosynthesis, which represents an additional link between glycolysis,PPP and biosynthesis. Metabolites have been suggested to function assignaling molecules. Examples include AMP, which is an allostericactivator for AMP-Activated Protein Kinase (AMPK), a kinase that sensesintracellular energy levels (ATP/AMP ratio), and glutamine, whichactivates leucine uptake, leading to mTOR activation. The cellularlevels of 3-PG and 2-PG, two intermediates in glycolysis, haveadditional regulatory impact on metabolic enzymes to affect cellmetabolism and consequently proliferation, which provides an example tosuggest that glycolytic metabolites could also serve as signalingmolecules to control cell metabolism and cellular responses. Moreover,findings herein also indicate a feedback mechanism by which the productlevels (2-PG) of a metabolic enzyme (PGAM1) can regulate its substratelevels (3-PG) by affecting an alternative enzyme (PHGDH) that isinvolved in production of this substrate.

Targeting PGAM1 by a PGAM1-derived inhibitory peptide or PGAM inhibitorMJE3 attenuates cancer cell proliferation. Studies herein suggest thatprotein expression and enzyme activity levels of PGAM1 are important forcancer cell proliferation and tumor growth. Certain PGM1 inhibitorsherein exhibits promising efficacy in treatment of xenograft nude micein vivo with minimal toxicity, as well as in diverse human cancer cellsand primary leukemia cells from human patients in vitro with no obviousoff target effect and minimal toxicity to human cells. Anti-PGAM1 is apromising therapy in clinical treatment of tumors that heavily rely onthe Warburg effect.

Y26 Phosphorylation Enhances PGAM1 Enzyme Activity by Promoting H11Phosphorylation.

Phospho-proteomics studies identified PGAM1 as Y26 phosphorylated incancer and leukemia cells (FIG. 13A). In vitro kinase assays whereconducted where active, recombinant FGFR1 (rFGFR1) phosphorylatedpurified, Flag-tagged recombinant PGAM1 (rPGAM1) (FIG. 13B) at Y26 (FIG.13B), which was accessed by a specific phospho-PGAM1 (pY26) antibody.Inhibition of FGFR1 by TKI258 treatment results in decreased PGAM1activity in the presence but not absence of cofactor2,3-bisphosphoglycerate (2,3-BPG)(FIG. 13C). PGAM1 is believed to beactivated upon binding of 2,3-BPG, which may “phosphorylate” PGAM1 athistidine 11 (H11) by transferring the C3 phosphate. Our mutationalanalysis revealed that in the presence of 2,3-BPG, rFGFR1 significantlyactivates rPGAM1 WT and control Y133F mutant but not Y26F mutant (FIG.13D). Structural studies revealed that Y26 is close to the cofactor2,3-BPG binding site (FIG. 13E), suggesting a potential mechanismwherein Y26 phosphorylation by FGFR1 may induce conformational change topromote cofactor binding. To test this hypothesis, active rFGFR1 wasincubated with purified, recombinant PGAM1 WT, Y26F or a control Y133Fmutant in an in vitro kinase assay, followed by incubation with acompetitive 2,3-BPG fluorescent analogue(8-hydroxy-1,3,6-pyrenetrisulfonate). The decrease in fluorescence (ex:362 nm, em: 520 nm) compared with buffer control was measured as 2,3-BPGbinding ability. Phosphorylation of PGAM1 WT or a control Y133F mutantby FGFR1 resulted in a significant increase in the amount of bound2,3-BPG analogue, whereas substitution of PGAM1 Y26 abolished enhancedbinding of cofactor in the presence of rFGFR1 (FIG. 13F). Moreover, aquantitative mass spectrometry based study (FIG. 13G) revealed that theH11 phosphorylation levels of Y26F mutant is significantly lowercompared to PGAM1 WT in an in vitro kinase assay using PGAM1 proteinsincubated with rFGFR1 in the presence of 2,3-BPG (FIG. 13G; left).Similar results were obtained (FIG. 13G; right) when using Flag-taggedmouse PGAM1 (mPGAM1) WT and Y26F from “rescue” H1299 cells with stableknockdown of endogenous human PGAM1 (hPGAM1) and rescue expression ofFlag-mPGAM1 WT or Y26F mutant. These results suggest that Y26phosphorylation enhances PGAM1 activity by promoting 2,3-BPG binding toPGAM1 and consequently H11 phosphorylation.

Methods of Use

The compounds and pharmaceutical compositions disclosed can be used toinhibit the PGAM pathway. Examples are listed for the use of anexemplary compound in treating head and neck cancer, lung cancer, andleukemia. Other cancers have also been shown to favor the PGAM pathway:lung cancer, Durany et al., Phosphoglycerate mutase,2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymesin lung, colon and liver carcinomas, 75:7 British Journal of Cancer969-977 (1997), breast cancer, Durany et al, Phosphoglycerate mutase,2,3-bisphosphoglycerate phosphatase, creatine kinase and enolaseactivity and isoenzymes in breast carcinoma, 82:1 British Journal ofCancer 20-27 (2000), liver cancer, Durany et al., Phosphoglyceratemutase, 2,3-bisphosphoglycerate phosphatase and enolase activity andisoenzymes in lung, colon and liver carcinomas, 75:7 British Journal ofCancer 969-977 (1997), colon cancer, Durany et al., Phosphoglyceratemutase, 2,3-bisphosphoglycerate phosphatase and enolase activity andisoenzymes in lung, colon and liver carcinomas, 75:7 British Journal ofCancer 969-977 (1997) and colorectal cancer. Teruyuki Usuba,Purification and Identification of Monoubiquitin-phosphoglycerate MutaseB Complex from Human Colorectal Cancer Tissues, 94:5 InternationalJournal of Cancer 662-668 (2001). See also, Glycolysis inhibition foranticancer treatment, 25:34 Oncogene 4633-4646 (2006). The compounds andpharmaceutical compositions disclosed herein may be prescribed topatients diagnosed with or suffering from any form of cancer as atreatment for their ailment.

It is further contemplated that these compounds and compositions may beused in the prevention of all forms of cancer. Pharmaceuticalcompositions disclosed can be prescribed to subjects at risk for cancerin order to lower the incidence rate of cancer. Since cancer survivalrates are higher when the disease is caught in the early stages, apreventive treatment will increase the likelihood of survival forsubjects not yet diagnosed with the disease.

Other diseases that involve regulation of the PGAM pathway and in whichuse of the compounds disclosed would be beneficial include musculardystrophy (and other muscle disorders) and glycogen storage disease. SeeClown et al., Plasma phosphoglycerate mutase as a marker of musculardystrophy, 65:2 Journal of the Neurological Sciences 201-210 (1984). Incertain embodiments, the disclosure contemplates methods of usingcompounds disclosed herein in the treatment or prevention of musculardystrophy or other glycogen storage diseases by administration to asubject in need thereof

Anthracene-9,10-Dione Derivatives

In certain embodiments, the anthracene-9,10-dione derivative is acompound of Formula I,

prodrug, ester, or salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each individually andindependently hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl,carbocyclyl, aryl, or heterocyclyl, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ are optionally substituted with one or more, the same ordifferent, R⁹;

R⁹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁹ is optionally substituted with one or more, thesame or different, R¹⁰;

R¹⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁹ is optionally substituted with one or more, thesame or different, R¹¹;

R¹¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R⁷ is hydroxyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R⁸ is hydroxyl.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino wherein R¹ is optionally substitutedwith one or more, the same or different, R⁹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring is optionally substituted with one or more, thesame or different R¹⁰.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with an alkyl,wherein the alkyl group is optionally substituted with one or more, thesame or different R¹¹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with a methyl,wherein the methyl group is optionally substituted with one or more, thesame or different R¹¹.

In some embodiments, the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position with a methyl,wherein the methyl group is substituted with one or more, the same ordifferent halogens.

In some embodiments the disclosure relates to compounds of Formula I orsalts thereof, wherein R¹ is amino and is substituted with an aryl ring,wherein the aryl ring substituted in the para position withtrifluoromethane.

In certain embodiments, the derivative is3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid or3,4-dihydroxy-9,10-dioxo-N-(4-(trifluoromethyl)phenyl)-9,10-dihydroanthracene-2-sulfonamideprodrug, ester, or salt thereof optionally substituted with one or more,the same or different, substituent(s).

Pharmaceutical Compositions

The compounds of the present disclosure can be administered to a subjecteither alone or as a part of a pharmaceutical composition.

This application claims as a novel pharmaceutical composition, all theclaimed compounds combined with one or more pharmaceutical agents, aswell as the combination of one or more pharmaceutical agents with anycompound in the family represented by Formula I. Pharmaceuticallyacceptable salts, solvates and hydrates of the compounds listed are alsouseful in the method of the disclosure and in pharmaceuticalcompositions of the disclosure.

The pharmaceutical compositions of the present disclosure can beadministered to subjects either orally, rectally, parenterally(intravenously, intramuscularly, or subcutaneously), intracistemally,intravaginally, intraperitoneally, intravesically, locally (powders,ointments, or drops), or as a buccal or nasal spray.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable (such as olive oil, sesame oil and viscoleo) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the surfactants.

These compositions may also contain adjuvants such as preserving,emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms be controlled by addition of any of various antibacterialand antifungal agents, example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example sugars, sodium chloride, and the like. Prolonged absorptionof the injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or: (a) fillersor extenders, as for example, starches, lactose, sucrose, glucose,mannitol and silicic acid, (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar and as high molecular weight polyethylene glycols, and thelike.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be used inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients. Controlled slow release formulations arealso preferred, including osmotic pumps and layered delivery systems.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, viscoleo, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Suspensions, in addition to the activecompounds, may contain suspending agents, as for example, ethoxylatediso-stearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite agar-agarand tragacanth, or mixtures of these substances, and the like.

Compositions for rectal administrations are preferably suppositorieswhich can be prepared by mixing the compounds of the present disclosurewith suitable nonirritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax, which are solid at ordinarytemperatures but liquid at body temperature and therefore, melt in therectum or vaginal cavity and release the active component.

Dosage forms for topical administration of a compound of this disclosureinclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, eye ointments, powders, and solutionsare also contemplated as being within the scope of this disclosure.

Pharmaceutical compositions disclosed herein can be in the form ofpharmaceutically acceptable salts, as generally described below. Somepreferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure can also form internalsalts, and such compounds are within the scope of the disclosure. When acompound contains a hydrogen-donating heteroatom (e.g. NH), salts arecontemplated to cover isomers formed by transfer of the hydrogen atom toa basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include the aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemisalts of acids and bases can also beformed, for example, hemisulphate and hemicalcium salts. For a review onsuitable salts, see Handbook of Pharmaceutical Salts: Properties,Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporatedherein by reference.

The compounds described herein can be administered in the form ofprodrugs. A prodrug can include a covalently bonded carrier whichreleases the active parent drug when administered to a mammaliansubject. Prodrugs can be prepared by modifying functional groups presentin the compounds in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent compounds.Prodrugs include, for example, compounds wherein a hydroxyl group isbonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl group. Examples of prodrugs include, butare not limited to, acetate, formate and benzoate derivatives of alcoholfunctional groups in the compounds. Examples of structuring a compoundas prodrugs can be found in the book of Testa and Caner, Hydrolysis inDrug and Prodrug Metabolism, Wiley (2006) hereby incorporated byreference. Typical prodrugs form the active metabolite by transformationof the prodrug by hydrolytic enzymes, the hydrolysis of amides, lactams,peptides, carboxylic acid esters, epoxides or the cleavage of esters ofinorganic acids.

Pharmaceutical compositions typically comprise an effective amount of acompound and a suitable pharmaceutical acceptable carrier. Thepreparations can be prepared in a manner known per se, which usuallyinvolves mixing the at least one compound according to the disclosurewith the one or more pharmaceutically acceptable carriers, and, ifdesired, in combination with other pharmaceutical active compounds, whennecessary under aseptic conditions. Reference is made to U.S. Pat. Nos.6,372,778, 6,369,086, 6,369,087 and 6,372,733 and the further referencesmentioned above, as well as to the standard handbooks, such as thelatest edition of Remington's Pharmaceutical Sciences. It is well knownthat ester prodrugs are readily degraded in the body to release thecorresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet. (2006)21(3):173-85, entitled “Human carboxylesterase isozymes: catalyticproperties and rational drug design.

Generally, for pharmaceutical use, the compounds can be formulated as apharmaceutical preparation comprising at least one compound and at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activecompounds.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and can be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which can be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe at least one compound of the disclosure e.g., about 10, 25, 50, 100,200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The compound will generally be administered in an“effective amount,” by which it is meant any amount of a compound that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thesubject per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the subject per day, which can beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen can be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the subject and the nature and severity of thedisease/symptoms to be treated. Reference is made to U.S. Pat. Nos.6,372,778, 6,369,086, 6,369,087 and 6,372,733 and the further referencesmentioned above, as well as to the standard handbooks, such as thelatest edition of Remington's Pharmaceutical Sciences.

Formulations containing one or more of the compounds described hereincan be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and can be administeredto an individual without causing undesirable biological side effects orunwanted interactions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of the coating composition whichcan include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations can be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (NewYork, Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy,” 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems,” 6thEdition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material can contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants.

Diluents, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants can be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules, or particles can also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, or preservatives.

The compositions described herein can be formulation for modified orcontrolled release. Examples of controlled release dosage forms includeextended release dosage forms, delayed release dosage forms, pulsatilerelease dosage forms, and combinations thereof.

The extended release formulations are generally prepared as diffusion orosmotic systems, for example, as described in “Remington—The science andpractice of pharmacy” (20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000). A diffusion system typically consists of twotypes of devices, a reservoir and a matrix, and is well known anddescribed in the art. The matrix devices are generally prepared bycompressing the drug with a slowly dissolving polymer carrier into atablet form. The three major types of materials used in the preparationof matrix devices are insoluble plastics, hydrophilic polymers, andfatty compounds. Plastic matrices include, but are not limited to,methyl acrylate-methyl methacrylate, polyvinyl chloride, andpolyethylene. Hydrophilic polymers include, but are not limited to,cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described as fullypolymerized copolymers of acrylic and methacrylic acid esters with a lowcontent of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename Eudragit®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the tradenamesEudragit®RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D andEudragit® RS30D are copolymers of acrylic and methacrylic esters with alow content of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEudragit® RL30D and 1:40 in Eudragit® RS30D. The mean molecular weightis about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred.The code designations RL (high permeability) and RS (low permeability)refer to the permeability properties of these agents. Eudragit® RL/RSmixtures are insoluble in water and in digestive fluids. However,multiparticulate systems formed to include the same are swellable andpermeable in aqueous solutions and digestive fluids.

The polymers described above such as Eudragit® RL/RS can be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems can be obtained,for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit®RS, and 10% Eudragit® RL and 90% Eudragit® RS. One skilled in the artwill recognize that other acrylic polymers can also be used, such as,for example, Eudragit® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In a congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

Delayed release formulations are created by coating a solid dosage formwith a polymer film, which is insoluble in the acidic environment of thestomach, and soluble in the neutral environment of the small intestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition can be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and can be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit®. (RohmPharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55(soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 andabove), Eudragit® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and Eudragits® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials can also be used. Multi-layer coatings usingdifferent polymers can also be applied.

The preferred coating weights for particular coating materials can bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition can include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates can also be used. Pigmentssuch as titanium dioxide can also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), can also beadded to the coating composition.

Alternatively, each dosage unit in the capsule can comprise a pluralityof drug-containing beads, granules or particles. As is known in the art,drug-containing “beads” refer to beads made with drug and one or moreexcipients or polymers. Drug-containing beads can be produced byapplying drug to an inert support, e.g., inert sugar beads coated withdrug or by creating a “core” comprising both drug and one or moreexcipients. As is also known, drug-containing “granules” and “particles”comprise drug particles that can or can not include one or moreadditional excipients or polymers. In contrast to drug-containing beads,granules and particles do not contain an inert support. Granulesgenerally comprise drug particles and require further processing.Generally, particles are smaller than granules, and are not furtherprocessed. Although beads, granules and particles can be formulated toprovide immediate release, beads and granules are generally employed toprovide delayed release.

Combination Therapies

The cancer treatments disclosed herein can be applied as a sole therapyor can involve, conventional surgery or radiotherapy or chemotherapy.Such chemotherapy can include one or more of the following categories ofanti-tumor agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulfan and nitrosoureas); antimetabolites (for exampleantifolates such as fluoropyrimidines like 5-fluorouracil andgemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinosideand hydroxyurea); antitumor antibiotics (for example anthracyclines likeadriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin,mitomycin-C, dactinomycin and mithramycin); antimitotic agents (forexample vinca alkaloids like vincristine, vinblastine, vindesine andvinorelbine and taxoids like taxol and taxotere); and topoisomeraseinhibitors (for example epipodophyllotoxins like etoposide andteniposide, amsacrine, topotecan and camptothecin); and proteosomeinhibitors (for example bortezomib [Velcade®]); and the agent anegrilide[Agrylin®]; and the agent alpha-interferon

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-Her2 antibody trastuzumab and the anti-epidermal growthfactor receptor (EGFR) antibody, cetuximab), farnesyl transferaseinhibitors, tyrosine kinase inhibitors and serine/threonine kinaseinhibitors, for example inhibitors of the epidermal growth factor familyfor example EGFR family tyrosine kinase inhibitors such as:N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib), and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033), for example inhibitors of the platelet-derived growth factorfamily and for example inhibitors of the hepatocyte growth factorfamily, for example inhibitors of phosphotidylinositol 3-kinase (PI3K)and for example inhibitors of mitogen activated protein kinase kinase(MEK1/2) and for example inhibitors of protein kinase B (PKB/Akt), forexample inhibitors of Src tyrosine kinase family and/or Abelson (AbI)tyrosine kinase family such as dasatinib (BMS-354825) and imatinibmesylate (Gleevec™); and any agents that modify STAT signalling;

(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™]) andcompounds that work by other mechanisms (for example linomide,inhibitors of integrin ocvβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as an anti-RAS antisense; and

(viii) immunotherapy approaches, including for example ex-vivo andin-vivo approaches to increase the immunogenicity of subject tumorcells, such as transfection with cytokines such as interleukin 2,interleukin 4 or granulocyte-macrophage colony stimulating factor,approaches to decrease T-cell anergy, approaches using transfectedimmune cells such as cytokine-transfected dendritic cells, approachesusing cytokine-transfected tumor cell lines and approaches usinganti-idiotypic antibodies, and approaches using the immunomodulatorydrugs thalidomide and lenalidomide [Revlimid®].

The combination therapy also contemplates use of the disclosedpharmaceutical compositions with radiation therapy or surgery, as analternative, or a supplement, to a second therapeutic orchemotherapeutic agent.

Antibodies

In certain embodiments, the disclosure relates to an antibody that bindsthe PGAM1 phospho-Y26 epitope. In certain embodiments the antibody is ahuman chimera or a humanized antibody that binds PGAM1 phospho-Y26epitope.

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods (U.S. Pat. No.4,816,567). Monoclonal antibodies may also be produced by generation ofhybridomas in accordance with known methods. Hybridomas formed in thismanner are then screened using standard methods, such as enzyme-linkedimmunosorbent assay (ELISA) and surface plasmon resonance analysis, toidentify one or more hybridomas that produce an antibody thatspecifically binds with a specified antigen. Any form of the specifiedantigen may be used as the immunogen, e.g., recombinant antigen,naturally occurring forms, any variants or fragments thereof, as well asantigenic peptide thereof.

One exemplary method of making antibodies includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, in U.S. Pat. No. 5,223,409.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. U.S. Pat. No.7,064,244.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized, deimmunized,chimeric, may be produced using recombinant DNA techniques known in theart. A variety of approaches for making chimeric antibodies have beendescribed. See, e.g., U.S. Pat. Nos. 4,816,567 and 4,816,397. Humanizedantibodies may also be produced, for example, using transgenic mice thatexpress human heavy and light chain genes, but are incapable ofexpressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies described herein (U.S. Pat. No.5,225,539). All of the CDRs of a particular human antibody may bereplaced with at least a portion of a non-human CDR, or only some of theCDRs may be replaced with non-human CDRs. It is only necessary toreplace the number of CDRs required for binding of the humanizedantibody to a predetermined antigen.

Humanized antibodies or fragments thereof can be generated by replacingsequences of the Fv variable domain that are not directly involved inantigen binding with equivalent sequences from human Fv variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; 5,859,205; and 6,407,213. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable domains from at least one of aheavy or light chain. Such nucleic acids may be obtained from ahybridoma producing an antibody against a predetermined target, asdescribed above, as well as from other sources. The recombinant DNAencoding the humanized antibody molecule can then be cloned into anappropriate expression vector.

In certain embodiments, a humanized antibody is optimized by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or backmutations. An antibodyor fragment thereof may also be modified by specific deletion of human Tcell epitopes or “deimmunization” by the methods disclosed in U.S. Pat.Nos. 7,125,689 and 7,264,806. Briefly, the heavy and light chainvariable domains of an antibody can be analyzed for peptides that bindto WIC Class II; these peptides represent potential T-cell epitopes. Fordetection of potential T-cell epitopes, a computer modeling approachtermed “peptide threading” can be applied, and in addition a database ofhuman WIC class II binding peptides can be searched for motifs presentin the VH and VL sequences. These motifs bind to any of the 18 major MHCclass II DR allotypes, and thus constitute potential T cell epitopes.Potential T-cell epitopes detected can be eliminated by substitutingsmall numbers of amino acid residues in the variable domains, orpreferably, by single amino acid substitutions. Typically, conservativesubstitutions are made. Often, but not exclusively, an amino acid commonto a position in human germline antibody sequences may be used. The VBASE directory provides a comprehensive directory of humanimmunoglobulin variable region sequences. These sequences can be used asa source of human sequence, e.g., for framework regions and CDRs.Consensus human framework regions can also be used, e.g., as describedin U.S. Pat. No. 6,300,064.

EXAMPLES Synthesis of PGMI-004A and Methyl-2-PG

Synthesis of PGMI-004A involves two steps. The first step was tosynthesize 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride. Inbrief, 3,4-dihydroxy-9,10-dioxo-2-anthracene sulfonic acid sodium salt(1.71 g, 5.0 mmol) was added chlorosulfonic acid (50 mL) and the mixturewas heated to 90-100° C. for 5 hours. After cooled to room temperature,the reaction solution was added to ice (200 g). After the ice wasmelted, the solution was extracted with dichloromethane (3×150 mL) andthe organic phase was combined and dried over sodium sulfate. Afterremoval of dichloromethane, the residue was subjected to silicachromatography to give product3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride as a brown solid(0.59 g, 35%). ¹H NMR (500.1 MHz) (Acetone-d₆) δ: 12.95 (s, 1H), 11.65(br., 1H), 8.35 (m, 2H), 8.21 (s, 1H), 7.98 (m, 2H). ¹³C NMR (125.8 MHz)(DMSO-d₆) δ: 189.7, 181.8, 152.9, 150.0, 136.6, 136.4, 135.6, 134.9,134.4, 128.2, 128.0, 124.4, 119.7, 117.6. MS calcd. for C14H7ClO6S[M-H]−, 337.0 (calcd.); 336.9 (found).

The second step was to synthesizeN-(4-trifluoromethyl)-penyl-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(PGMI-004A). 3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride(0.50 g, 1.47 mmol) in dichloromethane (20 mL) was added triethylamine(4.0 equiv.) and 4-(trifluoromethyl)aniline (2.0 equiv.) and the mixturewas stirred at room temperature overnight. After filtration, the solventwas evaporated and the resulting solid was subjected to silicachromatography to give productN-(4-trifluoromethyl)-penyl-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamideas a red solid (0.29 g, 42%). ¹H NMR (500.1 MHz) (DMSO-d₆) δ: 8.12 (m,3H), 7.83 (m, 2H), 7.58 (m, 2H), 7.28 (m, 2H). ¹³C NMR (125.8 MHz)(DMSO-d₆) δ: 189.8, 180.6, 154.3, 143.2, 136.5, 135.4, 135.2, 134.3,129.0, 128.1, 127.9, 127.8, 127.7, 127.6, 126.8, 124.8, 124.6, 124.5,123.1, 119.8, 118.5, 114.4. MS calcd. for C21H12F3O6S [M-H]−, 462.0(calcd.); 462.0 (found).

To synthesize methyl ester of D(+)-2-phosphoglyceric acid,D(+)2-Phosphoglyceric acid sodium salt (20 mg) was added hydrogenchloride in methanol (5 mL) and the mixture was stirred at roomtemperature for 5 h. Removal of the solvent under reduced pressure gaveproduct methyl ester of D(+)-2-phosphoglyceric acid quantitatively as awhite solid. ¹H NMR (500.1 MHz) (CD₃OD) δ: 4.72 (m, 1H), 3.87 (m, 2H),3.77 (s, 3H). 1H NMR (125.8 MHz) (CD₃OD) δ: 170.8, 76.7 (J=5.41 Hz),63.6 (J=5.79 Hz), 52.4. ³¹P NMR (202.4 MHz) (CD₃OD) δ: −0.946.

In Vitro PGAM1 and Enolase Assays.

An in vitro PGAM1 assay was performed as primary screening. PGAM1 enzymemix was prepared containing 100 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂, 1mM ADP, 0.2 mM NADH, 5 mg/ml recombinant PGAM1, 0.5 units/ml enolase,0.5 units/ml recombinant pyruvate kinase M1, and 0.1 units/mlrecombinant LDH. 3-PG was added last at the final concentration of 2 mMto initiate the reaction. The decrease in autofluorescence (ex:340 nm,em:460 nm) from oxidation of NADH was measured as PGAM1 activity. An invitro enolase assay as secondary screening was also performed. Enolaseenzyme mix was prepared containing 100 mM Tris-HCl, 100 mM KCl, 5 mMMgCl₂, 1 mM ADP, 0.2 mM NADH, 0.5 units/ml enolase, 0.5 units/mlrecombinant pyruvate kinase M1, and 0.1 units/ml recombinant LDH. 2-PGwas added last at the final concentration of 2 mM to initiate thereaction. The decrease in autofluorescence (ex:340 nm, em:460 nm) fromoxidation of NADH was measured as enolase activity. For K_(d)determination, 2 μM of human PGAM1 proteins were mixed with differentconcentrations of PGMI-004A (1-500 μM). The fluorescence intensity (Ex:280 nm, em: 350 nm) from Tryptophan were measured. Assay was carried outin Tris-HCl buffer (10 mM Tris, pH 7.4, 100 mM NaCl) containing 5% DMSO.

PGMI-004A-3-PG Competitive Binding Assay.

2 μM rPGAM1 was incubated with different concentrations of PGMI-004A(0-40 μM) and 3-PG (0-400 μM), and Tryptophan fluorescence (ex: 280 nm,em:350 nm) from rPGAM1 was measured in 100 mM Tris-HCl buffer.Fluorescence intensity without any treatment is presented as 1.0.

Thermal Melt Shift Assay.

In brief, thermal shift assay of compound-protein interaction wasperformed in 384-well PCR plates with various compound concentrationsand 200 μg/ml protein in a buffer solution (20 mM Tris-HCl, 100 mM NaCl,pH 7.4). SYPRO orange was used as a dye to monitor the fluorescencechange at 610 nm. Small molecules were dissolved in DMSO and added toprotein solution. Final DMSO concentration of solution is 1%.Dissociation constants (Kds) for protein-ligand interaction werecalculated using the method as described.

The fluorescence intensity data were fitted to Eq. (1) to obtain ΔHu,ΔCpu, and T_(m) by nonlinear regression using the program Prism:

$\begin{matrix}{{{{F(T)} = {{F({post})} + \frac{{F({pre})} - {F({post})}}{1 + {\exp \mspace{11mu} \left\{ {{{- \frac{\Delta \; {Hu}}{R}}\left( {\frac{1}{T} - \frac{1}{T_{m}}} \right)} + {\frac{\Delta \; {Cpu}}{R}\left\lbrack {{\ln \mspace{14mu} \left( \frac{T}{T_{m}} \right)} + \frac{T_{m}}{T} - 1} \right\rbrack}} \right\}}}}}{where}{{{F(T)}\text{:}\mspace{14mu} {fluorescence}\mspace{14mu} {intensity}\mspace{14mu} {at}\mspace{14mu} {temperature}\mspace{11mu} T};}\text{}{{T_{m}\text{:}\mspace{14mu} {midpoint}\mspace{14mu} {temperature}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {protein}\text{-}{unfolding}\mspace{14mu} {transition}};}{{{F({pre})}\mspace{14mu} {and}\mspace{14mu} F\; ({post})\text{:}\mspace{11mu} {pretransitional}\mspace{14mu} {and}\mspace{14mu} {posttransitional}\mspace{14mu} {fluorescence}\mspace{14mu} {intensities}};}\text{}{R\text{:}\mspace{14mu} {gas}\mspace{14mu} {constant}},{{\Delta \; {Hu}\text{:}\mspace{14mu} {enthalpy}\mspace{14mu} {of}\mspace{14mu} {protein}\mspace{14mu} {unfolding}}\;;}}\text{}{\Delta \; {Cpu}\text{:}\mspace{14mu} {heat}\mspace{14mu} {capacity}\mspace{14mu} {change}\mspace{14mu} {on}\mspace{14mu} {protein}\mspace{14mu} {{unfolding}\;.}}} & (1)\end{matrix}$

The thermal unfolding parameters for 6PGD and PGAM1 alone weredetermined from 4 control wells, each containing 0.2 mg/mL protein. Theaverage Tm, ΔHu, ΔCpu are 326.00 K (52.85° C.), 103.9 kcal/mol, and 4.5kcal/mol for 6PGD, 322.43 K (49.28° C.), 157.0 kcal/mol, and 10.3kcal/mol for PGAM1, respectively. To calculate the ligand-bindingaffinity at T_(m) and T, equation (2) and (3) were used:

$\begin{matrix}{{F(T)} = {{F({post})} + \frac{{F({pre})} - {F({post})}}{1 + {\exp \mspace{11mu} \left\{ {{{- \frac{\Delta \; {Hu}}{R}}\left( {\frac{1}{T} - \frac{1}{T_{m}}} \right)} + {\frac{\Delta \; {Cpu}}{R}\left\lbrack {{\ln \; \left( \frac{T}{T_{m}} \right)} + \frac{T_{m}}{T} - 1} \right\rbrack}} \right\}}}}} & (2) \\{{{{F(T)} = {{F({post})} + \frac{{F({pre})} - {F({post})}}{1 + {\exp \; \left\{ {{{- \frac{\Delta \; {Hu}}{R}}\left( {\frac{1}{T} - \frac{1}{T_{m}}} \right)} + {\frac{\Delta \; {Cpu}}{R}\left\lbrack {{\ln \mspace{11mu} \left( \frac{T}{T_{m}} \right)} + \frac{T_{m}}{T} - 1} \right\rbrack}} \right\}}}}}{Where}}{{{{{K_{L{({Tm})}}\text{:}\mspace{14mu} {ligand}\mspace{14mu} {association}\mspace{14mu} {constant}\mspace{14mu} {at}\mspace{11mu} T_{m}};}\left\lbrack L_{Tm} \right\rbrack}\text{:}\mspace{14mu} {free}\mspace{14mu} {ligand}\mspace{14mu} {concentration}\mspace{14mu} {at}\mspace{14mu} T_{m}};}{{K_{L{(T)}}\text{:}\mspace{14mu} {ligand}\mspace{14mu} {association}\mspace{14mu} {constant}\mspace{14mu} {at}\mspace{14mu} T\mspace{11mu} \left( {37{^\circ}\mspace{11mu} {C.\mspace{14mu} {was}}\mspace{14mu} {used}\mspace{14mu} {for}\mspace{14mu} {all}\mspace{14mu} {the}\mspace{14mu} {calculation}} \right)};}{{\Delta H}_{L{(T)}}\text{:}\mspace{14mu} {{van}'}t\mspace{14mu} {Hoff}\mspace{14mu} {enthalpy}\mspace{14mu} {of}\mspace{14mu} {binding}\mspace{14mu} {at}\mspace{14mu} {temperature}\mspace{14mu} {T.\mspace{20mu} {Here}}\mspace{14mu} {we}\mspace{14mu} {use}\mspace{14mu} \text{-}5\mspace{14mu} {kcal}\text{/}{mol}\mspace{14mu} {which}\mspace{14mu} {makes}\mspace{14mu} {the}\mspace{14mu} {calculated}\mspace{14mu} {binding}\mspace{14mu} {constants}\mspace{14mu} {closer}\mspace{14mu} {to}\mspace{14mu} {the}\mspace{14mu} {real}\mspace{14mu} {{values}.}}} & (3)\end{matrix}$

Thermal shift data for 6PGD at 2 mM 3PG and 0.2 mM 6-PG concentrationswere used to calculate the K_(d) since both of the shift Tm are around2-2.5° C. and comparable. The K_(d) for 3PG and 6-PG with 6PGD are460±40 μM and 37±3 μM. Thermal shift data for PGAM1 at 40 μM PGMI-004Awas used to calculate the K_(d), which is 9.4±2.0 μM.

Cell Proliferation and Viability Assays.

For leukemia cell proliferation assay, 10×10⁴ cells were seeded innon-tissue culture coated 6-well plate and incubated at 37° C. forindicated times. Cell numbers were counted by trypan blue exclusionunder a microscope (×40) at indicated times and the percentage of cellproliferation was determined by comparing PGAM1 knockdown cells topLKO.1 vector expressing cells. For cell viability assays of leukemiacells, 10×10⁴ cells were seeded in non-culture coated 6-well plate andincubated with PGMI-004A at 37° C. for indicated times. Cell viabilitywas determined by counting drug-treated cells compared to DMSO-treatedcontrol cells with trypan blue exclusion under a microscope (×40) and byusing CellTiter96 Aqueous One solution proliferation kit (Promega). Foradherent cell proliferation assay such as H1299 and MDA-MB231 cells,5×10⁴ cells were seeded in 6-well plate 24 h before the assay starts andwere cultured at 37° C. 24 h after seeding, cells were treated with 5 μMMethyl-2PG and incubated at 37° C. for indicated times. Cellproliferation was determined by the increase in cell number indicatedtimes after the treatment starts compared to that at the treatmentstarts for each cell line (T=0). Cell numbers were counted by trypanblue exclusion under a microscope (×40). For adherent cell viabilityassay with trypan blue exclusion, 5×10⁴ cells were seeded in 6-wellplate 24 h before the assay starts and were cultured at 37° C. 24 hafter seeding, cells were treated with PGMI-004A and incubated at 37° C.for indicated times. Cell viability was determined by countingdrug-treated cells compared to DMSO-treated control cells with trypanblue exclusion under a microscope (×40). For MTT cell viability assay ofadherent cells, 5×10³ cells were seeded in 96-well plate 24 h before theassay starts and were cultured at 37° C. 24 h after seeding, cells weretreated with PGMI-004A and incubated at 37° C. for 3 days. Cellviability was determined by using CellTiter96 Aqueous One solutionproliferation kit (Promega).

Xenograft Studies.

Nude mice (Athymic Nude-Foxn1^(nu), female 6-8-week-old, Harlan) weresubcutaneously injected with 10×10⁶ H1299 cells harboring empty vectoron the left flank, and cells with stable knockdown of endogenous hPGAM1on the right flank, respectively. The tumors were harvested and weighedat the experimental endpoint, and the masses of tumors (g) derived fromcells with and without stable knockdown of endogenous hPGAM1 in bothflanks of each mouse were compared. Statistical analyses were performedby comparison in relation to the control group with a two-tailed pairedStudent's t test. For drug evaluation of PGMI-004A using xenograft mice,the drug was administered by daily i.p. injection at a dose of 100 mg/kgfrom 6 days after subcutaneous injection of H1299 cells on right flankof each mouse. Tumor growth was recorded by measurement of twoperpendicular diameters of the tumors over a 3-week course using theformula 4π/3×(width/2)²×(length/2). The tumors were harvested andweighed at the experimental endpoint. The masses of tumors (g) treatedwith vehicle control (DMSO:PEG400:PBS at a ratio of 4:3:3) and PGMI-004Awere compared and the p values were determined by a two-tailed Student'st test.

Primary Tissue Samples from Human Patients with Leukemia and HealthyDonors.

Approval of use of human specimens was given by the Institutional ReviewBoard of Emory University School of Medicine. All clinical samples wereobtained with informed consent with approval by the Emory UniversityInstitutional Review Board. Clinical information for the patients wasobtained from the pathological files at Emory University Hospital underthe guidelines and with approval from the Institutional Review Board ofEmory University School of Medicine and according to the HealthInsurance Portability and Accountability Act. Only samples from patientsthat were not previously treated with chemotherapy or radiation therapywere used. Mononuclear cells (MNCs) were isolated from peripheral bloodand bone marrow samples from human leukemia patients or peripheral bloodsamples from healthy donors using lymphocyte separation medium(Cellgro). Cells were cultured in RPMI 1640 medium supplemented with 10%FBS and penicillin/streptomycin and incubated with increasingconcentrations of PGMI-004A for up to 72 or 120 hours.

Antibodies

Phospho-Tyr antibody pY99 and FGFR1 antibody were from Santa CruzBiotechnology, Santa Cruz, Calif.; PGAM1 antibody was from Novus;antibodies against GST and β-actin were from Sigma, St. Louis, Mo.Specific antibody against phospho-PGAM1 (p-Y26) was generated. Specificantibody against phospho-PGAM1 (p-Y26) was generated by CST.

RNAi

ShRNA construct for PGAM1 knockdown was purchased from Open Biosystems,Huntsville, Ala. The sequence of shRNA used for knockdown is as follows:5′-CCGGCAAGAACTTGAAGCCTATCAACTCGAGTTGATAGGCTTCAAGTTCTTGTTTTTT G-3′ (SEQID NO:1).

PGAM1 enzyme assay.

Murine PGAM1 was Flag-tagged by PCR and subcloned into pMSCV-neo derivedGateway destination vector as described previously (Chen et al., Blood,2005, 106, 328-337). For GST-tagged PGAM1 expression in mammalian cells,PGAM1 variants were subcloned into pDEST27 vector (Invitrogen, Carlsbad,Calif.). For His-tagged PGAM1 expression in bacterial cells, PGAM1 wassubcloned into pET53 vector (Novagen).

(His)₆-tagged PGAM1 proteins were purified by sonication of highexpressing BL21(DE3)pLysS cells obtained from a 250 mL culture subjectedto IPTG-induction for 4 h. Cell lysates were resolved by centrifugationand loaded onto a Ni-NTA column in 20 mM imidazole. After a step of 2×washing, the protein was eluted with 250 mM imidazole. Proteins weredesalted on a PD-10 column and the purification efficiency was examinedby Coomassie staining and western blotting.

PGAM1 activity was measured by multiple enzymes coupled assay. PGAM1enzyme mix containing 100 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂, 1 mM ADP,0.2 mM NADH, 5 mg/ml recombinant PGAM1, 0.5 units/ml enolase, 0.5units/ml recombinant pyruvate kinase M1, and 0.1 units/ml recombinantLDH was prepared. 3-PG was added last at a final concentration of 2 mMto initiate the reaction. The decrease in autofluorescence (ex:340 nm,em:460 nm) from oxidation of NADH was measured as PGAM1 activity.

Cellular Metabolites Extraction and Measurement.

Cellular metabolites were extracted and spectrophotometrically measuredas described previously with some modifications. To determine cellularconcentration of 2-PG and 3-PG, 100 μL of packed cell pellets werehomogenized in 1.5 ml of hypotonic lysis buffer (20 mM HEPES (pH 7.0), 5mM KCl, 1 mM MgCl₂, 5 mM DTT, and protease inhibitor cocktail). Thehomogenates were centrifuged in a cold room at 4° C. for 10 minutes atmaximum speed, and the supernatants were applied to Amicon Ultra tubeswith 10 KDa cut off filter (Millipore). The flow through containing themetabolites was used for the measurement. NADH, ADP, and MgCl₂ wereadded to the flow through to final concentrations of 0.14 mM, 1 mM, and50 mM, respectively. Recombinant LDH and PKM1 proteins were added tofinal concentrations of 5 μg/ml and 10 μg/ml, respectively. Recombinantenolase protein was added to a final concentration of 50 μg/ml tomeasure cellular 2-PG. Once the reaction was initiated by enolase, adecrease in absorbance at 340 nm from NADH oxidation was measured by aDU800 spectrophotometer (Beckman). After termination of the enolasereaction, recombinant PGAM1 protein was added to a final concentrationof 25 μg/ml and the decrease in absorbance at 340 nm was immediatelymonitored to measure cellular 3-PG. 100 μL of 2-PG and 3-PG (Sigma)diluted with 1.5 ml of hypotonic lysis buffer were used as thestandards.

Alternatively, perchloric acid was used to quench metabolism and theacid extracts from H1299 cell lysates were then neutralized with KOH(DeBerardinis et al., 2007), followed by enzymatic reactions using thefinal supernatant to determine the 3-PG and 2-PG levels. Anisotope-ratio based detection method was used to determine intracellular3-PG and 2-PG levels in cells. In brief, pure standards were derivatizedwith a trimethylsilyl donor (Tri-Sil, Pierce) and used to determine theretention times of 2-PG and 3-PG on GC/MS. These standards were alsoused to identify parent ions containing all three 2-PG and 3-PG carbonson the derivatized molecule (m/z 459 for both 2-PG and 3-PG), and togenerate algorithms for natural isotope abundance correction. Next, twodishes of H1299 cells were cultured in RPMI supplemented with[U-¹³C]glucose so that approximately 25% of the total glucose pool waslabeled. After 6 hours of culture, the cells were quickly rinsed inice-cold normal saline, lysed in 1 mL of cold 50% methanol, and frozenin liquid nitrogen. The lysates were freeze-thawed three times, thencentrifuged to remove debris. The supernatant from the first sample wasevaporated to dryness, derivatized in Tri-Sil, then injected (5 μL) ontoan Agilent 6890 GC networked to an Agilent 5973 Mass Selective Detector.This confirmed that the fractional enrichments of glucose, lactate, 2-PGand 3-PG were 25-30%, with only the unenriched and fully enrichedspecies present. The second sample was spiked with 25 nmoles ofunlabeled 2-PG and 3-PG, derivatized, and analyzed by GC/MS. The 2-PGand 3-PG enrichments in this sample were 5.8% and 6.3%, respectively(Response FIG. 1B; left). Using the factor of isotopic dilutionresulting from addition of 25 nmoles of unlabeled standards (4.8 for2-PG and 4.1 for 3-PG), the cellular lysate contained approximately 6.6nmoles of 2-PG and 8.1 nmoles of 3-PG. Assuming a 100 μL volume for thecell pellet, these figures correlate to intracellular concentrations of66 μM for 2-PG and 81 μM for 3-PG, very close to the values measured bythe enzymatic assay.

To determine cellular concentration of 6-PG, 200 μL of packed cellpellets were homogenized in 0.6 ml of hypotonic lysis buffer. Thehomogenates were centrifuged in a cold room at 4° C. for 10 minutes atmaximum speed, and the supernatants were applied to Amicon Ultra tubeswith 10 KDa cut off filter (Millipore). The flow through containing themetabolites was used for the measurement. Tris-HCl (pH8.1) and MgCl₂were added to the flow through to final concentrations of 50 mM and 1mM, respectively. Recombinant 6PGD protein was added to a finalconcentration of 10 μg/ml, and the reaction was initiated by adding NADP(final concentration of 0.1 mM) to the reaction mixture. An increase inabsorbance at 340 nm from NADPH production was measured by a DU800spectrophotometer (Beckman). 200 μL of 6-PG (Sigma) diluted with 0.6 mlof hypotonic lysis buffer were used as the standards.

¹⁴C-Lipid Synthesis and ¹⁴C-RNA Synthesis Assays.

¹⁴C-lipids synthesized from ¹⁴C-glucose were measured. Subconfluentcells seeded on a 6-well plate were pre-incubated with PGMI-004A for 2 hprior to the addition of ¹⁴C-glucose. Cells were then incubated incomplete medium spiked with 4 μCi/ml of D-[6-¹⁴C]-glucose for 2 h in thepresence of PGMI-004A, washed twice with PBS, and lipids were extractedby the addition of 500 μL hexane:isopropanol (3:2 v/v). Wells werewashed with an additional 500 μL of hexane:isopropanol solution, andextracts were combined and air dried with heat. Extracted lipids wereresuspended in 50 μL of chloroform, and subjected to scintillationcounting. Scintillation counts were normalized with cell numbers countedby a microscope (×40). ¹⁴C-RNA synthesized from ¹⁴C-glucose wasmeasured. Subconfluent cells seeded on a 6-well plate were pre-incubatedwith PGMI-004A for 2 h prior to the addition of ¹⁴C-glucose. Cells werethen incubated in complete medium spiked with 4 μCi/ml ofD-[U-¹⁴C]-glucose for 2 h in the presence of PGMI-004A. RNA was thenextracted using RNeasy columns (Qiagen) and ¹⁴C-RNA was assayed byscintillation counter. ¹⁴C counts for each sample were normalized by theamount of RNA.

¹⁴C-Serine Synthesis Assay.

Serine synthesis flux from ¹⁴C glucose was measured as describedpreviously (Parry, 1957) with modification. Sub-confluent cells seededon a 6 cm dish were incubated in complete medium spiked with 40 μCi/mlof D-[U-¹⁴C]-glucose overnight. The incubated cells were lysed withhypotonic lysis buffer (20 mM HEPES (pH 7.0), 5 mM KCl, 1 mM MgCl₂, 5 mMDTT, and protease inhibitor cocktail), and the cell lysates were spundown by centrifugation to remove cell debris. The supernatant containing100 μg of total protein was spotted onto Whatman 3MM paper.One-dimensional chromatogram was run in water saturated phenol, andtwo-dimensional chromatogram was run in 1-butanol:acetic acid:watersolution at the ratio of 100:22:50. L-serine (Sigma) was used as astandard. L-serine dissolved in hypotonic lysis buffer was spotted ontoanother paper, which was run under the same conditions in parallel withthe cell lysate sample. Spots of amino acids were developed by ninhydrinreaction. The spot corresponding to serine was identified based on R^(f)values of L-serine standard, cut out from the paper, and directlysubjected to scintillation counting.

Glycolytic Rate Assay.

Glycolytic rate was measured by monitoring the conversion of5-³H-glucose to ³H₂O. 10⁶ cells were washed once in PBS prior toincubation in 1 ml of Krebs buffer without glucose for 30 min at 37° C.The Krebs buffer was then replaced with Krebs buffer containing 10 mMglucose spiked with 10 μCi of 5-³H-glucose. Following incubation for 1 hat 37° C., triplicate 50 μl aliquots were transferred to uncapped PCRtubes containing 50 μl of 0.2 N HCl, and a tube was transferred into aneppendorf tube containing 0.5 ml of H₂O for diffusion. The tubes weresealed, and diffusion was allowed to proceed for a minimum of 24 h at34° C. The amounts of diffused ³H₂O were determined by scintillationcounting.

Lactate Production, Oxygen Consumption and Intracellular ATP Assays.

Cellular lactate production was measured under normoxia with afluorescence-based lactate assay kit (MBL). Phenol red-free RPMI mediumwithout FBS was added to a 6 well-plate of subconfluent cells, and wasincubated for 1 h at 37° C. After incubation, 1 ml of media from eachwell was assessed using the lactate assay kit. Cell numbers were countedby a microscope (×40). Oxygen consumption rates were measured with aClark type electrode equipped with 782 oxygen meter (StrathkelvinInstruments). 1×10⁷ cells were resuspended in RPMI 1640 medium with 10%FBS and placed into a water-jacked chamber RC300 (StrathkelvinInstruments) and recording was started immediately. Intracellular ATPconcentration was measured by an ATP bioluminescent somatic cell assaykit (Sigma). 1×10⁶ cells were trypsinized and resuspended in ultrapurewater. Luminescence was measured with spectrofluorometer (SPECTRA MaxGemini; Molecular Probe) immediately after the addition of ATP enzymemix to cell suspension.

NADPH/NADP⁺ Ratio Assay.

NADPH/NADP⁺ kit (BioAssay Systems) was used to measure cellularNADPH/NADP⁺ ratio. Subconfluent cells seeded on a 10 cm dish werecollected by a scraper, washed with PBS, and lysed with 200 μL of NADP⁺(or NADPH) extraction buffer. Heat extract was allowed to proceed for 5minutes at 60 degrees before adding 20 μL of assay buffer and 200 μL ofthe counter NADPH (or NADP⁺) extraction buffer to neutralize theextracts. The extracts were spun down and the supernatants were reactedwith working buffer according to the manufacturer's protocol. Theabsorbance at 565 nm from the reaction mixture was measured with platereader.

Oxidative PPP Flux Assay Using ¹⁴CO₂ Release.

Cells were seeded on 6-cm dishes that are placed in a 10-cm dish with 2sealed pinholes on the top. ¹⁴CO₂ released from cells was collected bycompletely sealing the 10-cm dish, in which the cells on the 6-cm dishwere incubated in 2 ml of medium containing [1-¹⁴C]- or [6-¹⁴C]-glucose,respectively, at a final specific activity of 10 μCi/ml glucose at 37°C. for 3 h. The oxidative PPP flux was stopped by injecting 0.3 ml of50% TCA through one of the holes on the top, and at the same time ¹⁴CO₂released was trapped by injecting 0.3 ml of Hyamine Hydroxide into asmall cup placed on the 10-cm dish through the second hole. Krebs cyclemeasurements, obtained in parallel samples incubated with[6-¹⁴C]-glucose, were used to correct the oxidative PPP fluxmeasurements obtained from samples incubated with [1-¹⁴C]-glucose. Eachdish was completely re-sealed with parafilm and incubated overnight atroom temperature. Hyamine Hydroxide in the small cup was dissolved into60% methanol and directly subjected to scintillation counting.

G6PD and 6PGD Assays.

G6PD activity was determined by the NADPH production rate from G6PD and6PGD, then subtracting that of 6PGD, since a product of G6PD,6-phosphogluconolactone, is rapidly hydrolyzed to a substrate of 6PGD,6-phosphogluconate, in cells. To obtain the combined dehydrogenaseactivity, substrates for both dehydrogenase enzymes were added to acuvette. In another cuvette, substrates for the second enzyme, 6PGD,were added to obtain the activity of this enzyme. Substrateconcentrations were as follows: 0.2 mM glucose 6-phosphate, 0.2 mM6-phosphogluconate, and 0.1 mM NADP⁺. 10 μg of cell lysates or 1 μg ofrecombinant protein were added to a cuvette containing buffer (50 mMTris, 1 mM MgCl₂, pH 8.1) and then the reaction was initiated by NADP⁺.The increase of absorbance at 341 nm was measured by a DU800spectrophotometer (Beckman).

PHGDH Enzyme Assay.

PHGDH activity was spectrophotometrically measured as describedpreviously. PHGDH enzyme buffer containing 200 mM Tris-HCl (pH8.1), 400mM KCl, 0.6 mM NAD, 2 mM GSH, 10 mM EDTA was mixed with cell lysates orrecombinant PHGDH protein. The reaction was initiated by the addition of3-PG to a final concentration of 10 mM and followed by measuring theincrease in absorbance at 340 nm over a 10 min period.

Primary Tissue Samples from Human Patients with Leukemia and HealthyDonors

Only samples from patients that were not previously treated withchemotherapy or radiation therapy were used. Mononuclear cells (MNCs)were isolated from peripheral blood and bone marrow samples from humanleukemia patients or peripheral blood samples from healthy donors usinglymphocyte separation medium (Cellgro). Cells were cultured in RPMI 1640medium supplemented with 10% FBS and penicillin/streptomycin andincubated with increasing concentrations of PGMI-004A for up to 72 or120 hours.

Xenograft Studies

An in vitro PGAM1 assay was used as primary screening followed by thesecondary screening using an in vitro enolase assay to exclude screenedcompounds with potential off target effect. In xenograft studies, nudemice were subcutaneously injected with 10×10⁶ H1299 cells stablyexpressing mPGAM1 WT and Y26F with stable knockdown of endogenous hPGAM1on the left and right flanks, respectively or 10×10⁶ H1299 cells withand without stable knockdown of endogenous hPGAM1 on the left and rightflanks, respectively. The tumors were harvested and weighed at theexperimental endpoint. Statistical analyses were done by comparison inrelation to the control group with a two-tailed paired Student's t test.For drug evaluation of PGMI-004A using xenograft mice, the drug wasadministered by daily i.p. injection at a dose of 100 mg/kg from 6 daysafter subcutaneous injection of H1299 cells on right flank of eachmouse. Tumor growth was recorded by measurement of two perpendiculardiameters of the tumors over a 3-week course using the formula4π/3×(width/2)²×(length/2).

Nude mice (Athymic Nude-Foxn1nu, female 6-8-week-old, Harlan) weresubcutaneously injected with 10×10⁶ H1299 cells harboring empty vectoror rescue cells stably expressing mPGAM1 WT on the left flank, and cellswith stable knockdown of endogenous hPGAM1 or rescue cells stablyexpressing mPGAM1 Y26F on the right flank, respectively. The tumors wereharvested and weighed at the experimental endpoint, and the masses oftumors (g) derived from cells expressing mPGAM WT or Y26F mutant in bothflanks of each mouse, or those of tumors derived from cells with andwithout stable knockdown of endogenous hPGAM1 in both flanks of eachmouse, were compared. Statistical analyses were performed by comparisonin relation to the control group with a two-tailed paired Student's ttest. For drug evaluation of PGMI-004A using xenograft mice, the drugwas administered by daily i.p. injection at a dose of 100 mg/kg from 6days after subcutaneous injection of H1299 cells on right flank of eachmouse. Tumor growth was recorded by measurement of two perpendiculardiameters of the tumors over a 3-week course using the formula4π/3×(width/2)2×(length/2). The tumors were harvested and weighed at theexperimental endpoint, and the masses of tumors (g) treated with vehiclecontrol (DMSO:PEG400:PBS at a ratio of 4:3:3) and PGMI-004A werecompared by a two-tailed unpaired Student's t test.

PGAM1 is Important for Cancer Cell Proliferation.

The effects of targeted down-regulation of diverse glycolytic enzymeswere examined by shRNA in cancer cells. A group of glycolytic enzymeswere found that are important to cancer cell metabolism andproliferation, including pyruvate kinase M2 (PKM2; 5) and PGAM1. In manycancers, including hepatocellular carcinoma and colorectal cancer, PGAM1activity is increased compared with normal tissues. PGAM1 geneexpression is believed to be up-regulated due to loss of TP53 in cancercells, as TP53 negatively regulates PGAM1 gene expression. As shown inFIG. 1, stable knockdown of PGAM1 in lung cancer H1299, head and neckcancer 212LN, breast cancer MDA-MB231 and leukemia KG1a, K562 and Molm14cells results in decreased cell proliferation with reduced PGAM1activity and lactate production. These results suggest a role of PGAM1protein levels in cancer cell proliferation and maintenance ofglycolysis.

Y26 Phosphorylation Activates PGAM1 by Promoting H11 Phosphorylation.

Phospho-proteomics studies identified PGAM1 as phosphorylated atmultiple tyrosine sites including Y26, Y92, Y119 and Y133 in murinehaematopoietic Ba/F3 cells expressing constitutively active ZNF198-FGFR1fusion tyrosine kinase 5. This was confirmed in an in vitro kinase assaywhere active, recombinant FGFR1 (rFGFR1) directly phosphorylatedpurified, Flag-tagged recombinant PGAM1 (rPGAM1) at tyrosine residues.In addition, GST-tagged PGAM1 was tyrosine phosphorylated in COS7 cellstransiently co-transfected with plasmids encoding FGFR1 wild type (WT),but not in cells co-expressing GST-PGAM1 and a kinase dead (KD) form ofFGFR1.

PGAM1 is believed to be activated upon binding of cofactor2,3-bisphosphoglycerate (2,3-BPG), which was suggested to“phosphorylate” PGAM1 at histidine 11 (H11) by transferring the C3phosphate to H11. In a PGAM1 enzyme activity assay, incubation withcofactor, 2,3-BPG significantly activates PGAM1 in cell lysates fromFGFR1-expressing lung cancer H1299 and leukemia KG1a cells, andtreatment with the FGFR1 small molecule inhibitor TKI258 significantlydecreases PGAM1 enzyme activity, only in the presence but not absence of2,3-BPG. In consonance with this observation, in a PGAM1 enzyme assaycoupled with the FGFR1 in vitro kinase assay, rFGFR1 significantlyactivated rPGAM1 enzyme activity only in the presence but not absence of2,3-BPG. Mutational analysis provided that, in the absence of 2,3-BPG,tyrosine phosphorylation by FGFR1 did not alter enzyme activity ofrPGAM1 WT, Y26F, Y119F or Y133F.

Interestingly, Y92F mutant lost PGAM1 enzyme activity in the presenceand absence of rFGFR1, suggesting that Y92 is intrinsically required forPGAM1 enzyme activity. In contrast, in the presence of 2,3-BPG, rFGFR1significantly activates rPGAM1 Y119F and Y133F, in addition to rPGAM1 WTas previously observed. However, substitution of Y26 abolished theFGFR1-dependent increase in the PGAM1 enzyme activity. Y92F mutant againshowed a very low level of PGAM1 activity, however, incubation withrFGFR1 resulted in significantly increased PGAM1 enzyme activity of Y92Fin the presence of 2,3-BPG. These data together suggest that Y26phosphorylation is responsible for mediating FGFR1-dependent activationof PGAM1 in the presence of 2,3-BPG.

Structural studies revealed that both H11 and Y92 are directly proximalto the active site where both cofactor (2,3-BPG) and substrate (3-PG)bind, suggesting that Y92 is important for 2,3-BPG binding and PGAM1activity, consistent with our observation that substitution of Y92abolishes PGAM1 enzyme activity (FIG. 2d-2e ). This is also consistentwith a previous report that S14, T23, G24, R90, Y92, K99, R116 and R117are involved in binding of cofactor 2,3-BPG and substrate 3-PG, whichshare the same binding pocket on PGAM1.

Y26 is also close to the cofactor binding site; since FGFR1phosphorylates Y26 to activate PGAM1 in the presence of 2,3-BPG, thissuggests a potential mechanism wherein Y26 phosphorylation by FGFR1 mayinduce conformational change to promote cofactor binding. To test thishypothesis, active rFGFR1 was incubated with purified, recombinant PGAM1WT, Y26F or control Y133F mutant in an in vitro kinase assay, followedby incubation with 2,3-BPG fluorescent analogue(8-hydroxy-1,3,6-pyrenetrisulfonate). The decrease in fluorescence (ex:362 nm, em: 520 nm) compared with buffer control was measured as 2,3-BPGbinding ability.

Phosphorylation of PGAM1 WT or Y133F mutant by FGFR1 resulted in asignificant increase in the amount of bound 2,3-BPG analogue, whereassubstitution of PGAM1 Y26 abolished enhanced binding of cofactor in thepresence of rFGFR1.

Moreover, a quantitative mass spectrometry based study revealed that theH11 phosphorylation levels of Y26F mutant is significantly lowercompared to PGAM1 WT in an in vitro kinase assay using PGAM1 proteinsincubated with rFGFR1 in the presence of 2,3-BPG. Similar results wereobtained when using Flag-tagged mouse PGAM1 (mPGAM1) WT and Y26F from“rescue” H1299 cells with stable knockdown of endogenous human PGAM1(hPGAM1) and rescue expression of Flag-mPGAM1 WT or Y26F mutant,respectively. These results suggest that Y26 phosphorylation enhancesPGAM1 activity by promoting 2,3-BPG binding to PGAM1 and consequentlyH11 phosphorylation.

An antibody was generated that specifically recognizes PGAM1phospho-Y26. PGAM1 was phosphorylated at Y26 by rFGFR1 in vitro;inhibiting FGFR1 decreased PGAM1 Y26 phosphorylation in H1299 lungcancer cells and KG1a leukemia cells. In addition, FLT3 and JAK2 alsodirectly phosphorylated PGAM1 at Y26 in vitro, and inhibition ofFLT3-ITD by TKI258 and JAK2 by AG490 resulted in decreased Y26phosphorylation of PGAM1 in the pertinent human cancer cell lines. Itwas found that PGAM1 is commonly expressed and phosphorylated at Y26 indiverse leukemia and multiple myeloma cells associated with variousconstitutively activated tyrosine kinase mutants, as well as varioushuman solid tumor cells including lung, prostate, breast and head andneck cancer cells.

Y26 Phosphorylation of PGAM1 Contributes to Control of 3-PG and 2-PGLevels, Promoting Cancer Cell Metabolism, Proliferation and TumorDevelopment.

A specific phospho-PGAM1 antibody (pY26) was developed and used toidentify that PGAM1 is commonly expressed and phosphorylated at Y26 indiverse human leukemia cell lines associated with distinct LTKs (FIG.13A). Indeed, FGFR1 (FIG. 13B), JAK2 (FIG. 13C) and FLT3 (data notshown) directly phosphorylated PGAM1 at Y26 in vitro, and inhibition ofFGFR1 by TKI258 and JAK2 by AG490 resulted in decreased Y26phosphorylation of PGAM1 in the pertinent human cancer cell lines.“Rescue” H1299 were next generated cells by RNAi-mediated stableknockdown of endogenous hPGAM1 and rescue expression of Flag-taggedmPGAM1 WT or less active Y26F mutant (FIG. 13D). Compared with themPGAM1 WT rescue cells, Y26F cells have increased 3-PG (FIG. 13E; left)and decreased 2-PG (FIG. 13E; right) levels, as well as reduced cellproliferation (FIG. 13F), while treatment with methyl-2-PG significantlyrescues these phenotypes. Y26F cells also demonstrated attenuated tumorgrowth potential in xenograft nude mice compared to control mPGAM1 WTrescue cells (FIG. 13G). These data together suggest that PGAM1 Y26phosphorylation levels are important to control intracellular 3-PG and2-PG levels, which confers both metabolic and proliferative advantagesto cancer cells, representing an additional, acute regulatory mechanismunderlying PGAM1 upregulation in cancer cells.

PGAM1 Controls Intracellular 3-PG and 2-PG Levels, and is Important forGlycolysis and Anabolic Biosynthesis in Cancer Cells, as Well as CancerCell Proliferation and Tumor Growth

To better understand how cancer cells coordinate glycolysis and anabolicbiosynthesis, the effects of targeted downregulation of the glycolyticenzyme PGAM1 was examined. Stable knockdown of PGAM1 in lung cancerH1299, breast cancer MDA-MB231, acute myeloid leukemia Molm14 and headand neck cancer 212LN cells resulted in decreased PGAM1 activity. GlobalMetabolic Profiling (Metabolon) was performed using cell lysate samplesof parental H1299 cells and cells with stable knockdown of PGAM1. Theresults indicate that PGAM1 knockdown results in altered intracellularconcentrations of 118 biochemicals (61 upregulated and 57 downregulated)with p<0.05 using Welch's Two Sample t-tests. Among these biochemicals,the PGAM1 substrate 3-PG levels are increased in PGAM1 knockdowncompared to control cells. In consonance with this observation,attenuation of PGAM1 by shRNA in diverse cancer cells leads to not onlyincreased 3-PG (FIG. 1A) but also decreased 2-PG (FIG. 1B) levelscompared to corresponding control cells harboring an empty vector. Theintracellular levels of 3-PG and 2-PG determined using different methodsare comparable. In addition, stable overexpression of PGAM1 in 3T3 cellsresults in increased 2-PG and decreased 3-PG levels, compared to controlparental 3T3 cells. These results suggest a role for PGAM1 controllingthe metabolite levels of its substrate 3-PG and product 2-PG in cancercells.

The role of PGAM1 in cancer cell metabolism was examined. Compared tovector control cells, stable knockdown of PGAM1 results in decreasedglycolytic rate (FIG. 1C) and lactate production (FIG. 1D), as well asreduced glucose-dependent biosynthesis of RNA and lipids, accompanied byreduced NADPH/NADP+ ratio (FIG. 1E-1G, respectively). Since the PPPproduces NADPH and R5P to contribute to macromolecular biosynthesis,whether PGAM1 contributes to PPP flux was examined. Indeed, oxidativePPP flux is reduced in PGAM1 knockdown compared to control vector cells(FIG. 1H). Interestingly, attenuation of PGAM1 in cancer cells does notaffect glucose uptake rate, or intracellular ATP levels (FIG. 1I) or 02consumption rate (FIG. 1J) in either the presence or absence of ATPsynthase inhibitor oligomycin. These results suggest that downregulationof PGAM1 attenuates glycolysis, PPP and biosynthesis, but does notsignificantly affect glucose uptake or intracellular ATP levels.

In addition, stable knockdown of PGAM1 results in decreased cellproliferation in diverse human cancer and leukemia cells (FIG. 1K).Moreover, a xenograft experiment was performed in which nude mice weresubcutaneously injected with control H1299 cells harboring an emptyvector on the left flank and PGAM1 knockdown H1299 cells on the rightflank (FIG. 1L; left). The mice were monitored for tumor growth over a6-week time period. The masses of tumors derived from PGAM1 knockdownH1299 cells were significantly reduced compared to those of tumorsformed by vector control cells (FIG. 1L; right).

PGAM1 Knockdown Results in Elevated Levels of 3-PG, which Binds to andInhibits 6PGD by Competing with its Substrate 6-Phosphogluconate (6-PG)

The molecular mechanism by which PGAM1 regulates the PPP was explored.Experiments suggests that the abnormally high levels of 3-PG in PGAM1knockdown cells may account for inhibition of oxidative PPP flux (FIG.1). To test this hypothesis, the effect of 3-PG was examined onglucose-6-phosphate dehydrogenase (G6PD), the first and most importantenzyme of the oxidative PPP, which produces NADPH, and6-phosphogluconate dehydrogenase (6PGD), an enzyme that also producesNADPH while converting 6-phosphogluconate into ribulose 5-phosphate inthe presence of NADP+. In vitro 6PGD and G6PD assays were performed inthe presence of increasing concentrations of 3-PG. Physiologicalconcentrations of 3-PG in human cells are reported to be approximately50-80 μM. In H1299, MDA-MB231 and Molm14 cells, the 3-PG levels areapproximately 60-80 μM in control vector cells and 200-300 μM in PGAM1knockdown cells, while the 3-PG concentrations are approximately 160 μMand 310 μM in 212LN control and PGAM1 knockdown cells, respectively.Thus, the effects of increasing concentrations of 3-PG on G6PD and 6PGDenzyme activities was examined according to the aforementionedphysiological 3-PG levels in tumor cells.

Treatment with 3-PG concentrations analogous to those in PGAM1 knockdownH1299 cells (˜250 μM) results in decreased enzyme activity of 6PGD (FIG.2A) in H1299 cell lysates or recombinant 6PGD (r6PGD) (FIG. 2B), whereasthe physiological 3-PG concentrations determined in control H1299 cells(˜60 μM) do not significantly affect 6PGD enzyme activity in bothexperiments. In control experiments, treatment with increasingconcentrations of 3-PG did not significantly affect G6PD activity inH1299 cell lysates or rG6PD activity. In addition, 2-PG did not affect6PGD enzyme activity in H1299 cell lysates or r6PGD activity. Theseresults suggest that abnormally high levels of 3-PG, as in PGAM1knockdown cells, may selectively and directly inhibit 6PGD but not G6PD.

To examine whether 3-PG binds to and inhibits 6PGD, a thermal melt shiftassay was performed to examine the interaction of protein (6PGD) and“ligand” (3-PG). Incubation of increasing concentrations of 3-PG raises6PGD melting temperature (Tm) in a dose-dependent manner, suggestingthat 3-PG directly binds to the protein (FIG. 2C). The Kd value forprotein-“ligand” interaction was calculated to be 460±40 μM. Moreover,kinetics studies were performed on the inhibition of 6PGD by 3-PG. Asshown in FIG. 2D, the Dixon plot indicates that 3-PG binds and inhibits6PGD. The inhibition constant (Ki) was determined to be 489±13 μM, inagreement with the Kd determined.

The intracellular concentration of 6-PG in H1299, MDA-MB231 and 212LNcells were determined to be 34.9±2.1 μM, 37.6±0.7 μM and 24.9±0.4 μM,respectively. Additional enzyme kinetics assays were performed to testwhether 3-PG at a concentration analogous to that in PGAM1 knockdownH1299 cells (˜250 μM) functions as a competitive or non-competitiveinhibitor of 6PGD in the presence of physiological concentrations of6-PG (˜35 μM). As shown in FIG. 2E, the Lineweaver-Burk plotdemonstrates that 3-PG functions as a competitive inhibitor of 6PGD.Since the Kd value for protein (6PGD)-ligand (6-PG) interaction wascalculated to be 37±3 μM in a thermal melt shift assay (FIG. 2F), thesedata together suggest that at physiological concentrations, 3-PG (˜60-80μM) cannot effectively compete with 6-PG (˜35 μM) to inhibit 6PGD incancer cells; however, upon attenuation of PGAM1, elevated cellular 3-PGlevels (˜250-300 μM) result in reduced 6PGD enzyme activity.

To further understand the structural properties of 3-PG mediatedinhibition of 6PGD, the apo-form of 6PGD (1.39 Å) was crystallizedsoaked with 3-PG to obtain the 3-PG-bound form of 6PGD (1.53 Å). TheFo-Fc density analysis revealed that the electron density of 3-PG waslocated in the active site of the 3-PG-bound 6PGD structure but not inthe apo-6PGD structure. 3-PG interacts with several residues (Y191,T262, R287, R446) in the active site of 6PGD that are important forsubstrate binding and enzymatic activity of 6PGD. Differentconformations were observed for Arg 446 and His 452 in the 3-PG-bound6PGD structure compared to the apo-form 6PGD structure. An alignment ofthree different 6PGD structures with bound NADP, 6-PG and 3-PG shows anoverlap of 3-PG and 6-PG in the active site. Together, these resultsdemonstrate that 3-PG directly binds to 6PGD and inhibits 6PGD enzymeactivity by competing with the cognate substrate 6-PG, representing amolecular mechanism to explain how PGAM1, as a glycolytic enzyme,contributes to the regulation of the oxidative PPP and consequentlyanabolic biosynthesis.

Rescue of Reduced 2-PG Levels in PGAM1 Knockdown Cells Results inDecreased 3-PG Levels by Activating 3-Phosphoglycerate Dehydrogenase(PHGDH)

In order to examine the effect of decreased 2-PG levels on cancer cellmetabolism, the aforementioned PGAM1 knockdown cancer cells were treatedwith a cell permeable agent, methyl-2-PG, which converts to 2-PG incells. In diverse PGAM1 knockdown cancer cells, treatment withmethyl-2-PG results in increased 2-PG cellular levels comparable tothose in the corresponding control vector cells (FIG. 4A). Methyl-2-PGtreatment rescues the reduced lactate production (FIG. 4B) but has nosignificant effect on intracellular ATP levels in H1299 cells withstable knockdown of PGAM1 compared to control vector cells. This resultsuggests that rescuing cellular 2-PG levels reverses the inhibitoryeffect of PGAM1 knockdown on glycolysis and allows downstream glycolyticreactions to resume and ultimately produce lactate. However, suchrescued glycolytic activity does not affect ATP levels, which isconsistent with our previous observation (FIG. 1I-1J).

Surprisingly, methyl-2-PG treatment rescues the decreased oxidative PPPflux and biosynthesis of RNA and lipids, as well as partially restoresthe reduced cell proliferation in H1299 PGAM1 knockdown cancer cellscompared to the corresponding control vector cells (FIG. 4C-FIG. 4F).Similar results were obtained using MDA-MB231 vector and PGAM1 knockdowncells. These data suggest that the increased 2-PG levels in PGAM1knockdown cells provide a feedback mechanism to rescue the abrogated PPPand anabolic biosynthesis upstream of PGAM1.

This hypothesis was tested by examining the effect of rescued 2-PGlevels on 3-PG concentrations in PGAM1 knockdown cells. Treatment withmethyl-2-PG results in decreased 3-PG concentrations in diverse PGAM1knockdown cells to levels that are comparable to the 3-PG concentrationsin the corresponding control vector cells (FIG. 5A). These resultsfurther suggest that PGAM1 controls 2-PG levels in cancer cells, whichcontributes to PGAM1-dependent coordination of glycolysis and anabolicbiosynthesis by adjusting 3-PG levels.

The molecular mechanism underlying 2-PG dependent feedback regulation ofintracellular 3-PG levels was evaluated. Besides conversion to 2-PGcatalyzed by PGAM1 in glycolysis, 3-PG also serves as a precursor forserine synthesis and can be converted to 3-phosphohydroxypyruvate (pPYR)by PHGDH. Since PGAM1 activity is attenuated in PGAM1 knockdown cells,it is possible that the rescued cellular 2-PG levels by methyl-2-PGtreatment decreases 3-PG levels by activating PHGDH. This hypothesis wastested by examining the effect of 2-PG on PHGDH activity. PGAM1knockdown cells were used to exclude the endogenous PGAM1 effect on 3-PGand 2-PG in the PHGDH enzyme activity reactions. Indeed, treatment with2-PG concentrations equivalent to those determined in control H1299cells (˜45 μM) or methyl-2-PG treated PGAM1 knockdown cells (˜60 μM)results in higher PHGDH enzyme activity in H1299 PGAM1 knockdown celllysates (FIG. 5B; left). Similar results were obtained by treating 212LNPGAM1 knockdown cell lysates with increasing concentrations of 2-PG(FIG. 5B; right). Moreover, treatment with increasing concentrations of2-PG results in increased enzyme activity of recombinant PHGDH (rPHGDH)(FIG. 5C). In contrast, 2-PG concentrations that correspond to thosedetermined in PGAM1 knockdown cells (˜15 μM) did not significantlyaffect PHGDH activity. Together, these studies reveal a feedbackmechanism by which cellular 2-PG levels contribute to control of 3-PGlevels in cells through regulation of PHGDH.

In addition, stable knockdown of PGAM1 results in significantlydecreased serine biosynthesis, while treatment with methyl-2-PG rescuesthe phenotype (FIG. 5D). Moreover, shRNA-mediated knockdown of PHGDH(FIG. 5E) does not affect rescued 2-PG levels in PGAM1 knockdown cellsupon treatment with methyl-2-PG, while PHGDH knockdown abolishes themethyl-2-PG dependent decrease of the elevated 3-PG levels in H1299PGAM1 knockdown cells (FIG. 5F, left and right, respectively). Thesedata support the hypothesis that PGAM1 controls 2-PG levels to regulatePHGDH, which consequently regulates 3-PG levels by diverting 3-PG inserine biosynthesis. Furthermore, knockdown of PHGDH in PGAM1 stableknockdown cells reverses the methyl-2-PG treatment dependent rescue ofoxidative PPP flux as well as biosynthesis of serine, lipids and RNA(FIG. 5G-5H, respectively). These data together suggest that, besidesbeing a glycolytic metabolite, 2-PG may also signal through PHGDH toprovide regulation of PPP flux and anabolic biosynthesis, at least inpart by regulating 3-PG levels.

PGAM1 Enzyme Activity Strikes a Balance Between 3-PG and 2-PG Levels,which Coordinates Glycolysis and Biosynthesis to Promote Cancer CellProliferation

In order to study the role of PGAM1 enzyme activity in cancer metabolismand tumor development, a small molecule inhibitor of PGAM1 wasidentified. Currently the only reported PGAM1 inhibitor is MJE3, whichspecifically inhibits PGAM1 activity exclusively in intact cells,probably by targeting the active site of PGAM1 with certainmodifications in vivo (Evans et al., 2007; Evans et al., 2005). Ascreening strategy was designed using coupled PGAM1 and enolase assaysand identified three lead small molecule compounds, including alizarin,as PGAM1 inhibitors from a library of FDA approved 2,000 small moleculecompounds (FIG. 6A). 1,2-Dihydroxyanthraquinone a.k.a. alizarin(C14H804) (FIG. 6B; top) is a prominent dye, originally derived from theroots of plants of the Madder genus. Treatment with alizarin results indecreased proliferation of human leukemia KG1a cells in a dose-dependentmanner.

Alizarin Red S (FIG. 6B; middle) was identified as a more potent PGAM1inhibitor from a group of alizarin derivatives. Alizarin Red Sderivatives were designed by adding hydrophobic groups through asulfonamide bond. Among these compounds, PGAM1 inhibitor 004A(PGMI-004A)(FIG. 6B; bottom), is less potent than Red S; however, invitro, it demonstrates enhanced potency to inhibit PGAM1 in leukemiaKG1a cells compared to its parental compounds. This may be due to thefact that PGMI-004A is more hydrophobic than alizarin and alizarin RedS, which confers better cell permeability.

PGMI-004A inhibits PGAM1 with an IC50 of approximately 13.1 μM (FIG. 6C)and the K_(d) value of the PGMI-004A-PGAM1 interaction was determined tobe 7.2±0.7 μM from fluorescence-based binding assay (FIG. 6D). In acompetitive binding assay where PGMI-004A was incubated with recombinantPGAM1 proteins in the presence of different concentrations of PGAM1substrate 3-PG, increasing concentrations of 3-PG caused an increase inthe fluorescence intensity from PGMI-004A-unbound form of PGAM1 in thepresence of different concentrations of PGMI-004A, but not in theabsence of PGMI-004A (FIG. 6E). This suggests that PGMI-004A mayallosterically modulate the enzyme activity of PGAM1. The Ki value wasdetermined to be 3.91±2.50 μM using Dixon plot analysis (FIG. 6F). Inaddition, a thermal melt shift assay was performed to examine theinteraction of protein (PGAM1) and ligand (PGMI-004A). Incubation ofincreasing concentrations of PGMI-004A raises PGAM1 melting temperature(Tm) in a dose-dependent manner, suggesting that PGMI-004A directlybinds to the protein (FIG. 6G). The K_(d) value for protein-ligandinteraction was calculated to be 9.4±2.0 μM. Together, these resultssuggest that PGMI-004A directly binds to PGAM1 and inhibits its enzymeactivity.

Inhibition of PGAM1 activity by PGMI-004A treatment results in decreased2-PG and increased 3-PG levels in H1299 cells, which could be rescued bytreatment with methyl-2-PG (FIG. 7A). Moreover, treatment with PGMI-004Aresults in significantly reduced lactate production that was rescued bymethyl-2-PG treatment (FIG. 7B), but has no significant effect onintracellular ATP levels (FIG. 7C). In consonance with theseobservations, the rescued lactate production due to methyl-2-PGtreatment was abolished when enolase was knocked down or inhibited byspecific inhibitor NaF in PGMI-004A treated cells. These results alsosuggest that rescued 2-PG derived from methyl-2-PG is metabolized bycells to restore the decreased glycolysis due to PGAM1 inhibition incancer cells. PGMI-004A treatment results in decreased oxidative PPPflux (FIG. 7D) and NADPH/NADP+ ratio (FIG. 7E), as well as reducedbiosynthesis of lipids and RNA (FIG. 7F-7G, respectively) and cellproliferation (FIG. 7H) in H1299 cells. These phenotypes are similar tothose observed in PGAM1 knockdown cells, which could be significantlyrescued by treatment with methyl-2-PG, suggesting that PGMI-004A targetsPGAM1 to inhibit cancer cell metabolism and proliferation.

PGMI-004A treatment results in decreased cell proliferation of diversehuman cancer and leukemia cells (FIG. 7I-7J; FIG. 10E-10H), but notcontrol human dermal fibroblasts (HDF), human foreskin fibroblasts(HFF), human HaCaT keratinocyte cells and human melanocyte PIG1 cells(FIGS. 7K and 10I), suggesting minimal non-specific toxicity ofPGMI-004A in normal, proliferating human cells.

Targeting PGAM1 by PGMI-004A Treatment Inhibits Cancer CellProliferation and Tumor Growth, and Alters 3-PG and 2-PG Levels inPrimary Leukemia Cells from Human Patients, Leading to AttenuatedLeukemia Cell Proliferation

An in vivo drug treatment experiment was performed. Initial toxicitystudies by chronic injection of PGMI-004A to nude mice for 4 weeksrevealed that 100 mg/kg/day administered intraperitoneally is awell-tolerated dose. In addition, continuous treatment with PGMI-004A(100 mg/kg/day) for 7 days did not result in significant alteration inbody weight, complete blood counts (CBC) or hematopoietic properties ofnude mice. Histopathological analyses revealed that no notabledifferences between the vehicle-treated and PGMI-004A-treated groupswere evident. Xenograft experiments were performed by injecting H1299cells to nude mice. Six days post-injection, mice were divided into twogroups (n=8/group) and treated with either PGMI-004A (100 mg/kg/day) orvehicle for 21 days. PGMI-004A treatment results in significantlydecreased tumor growth and tumor size in treated mice compared with micereceiving vehicle control (FIG. 8A-8B, respectively). Moreover,treatment with PGMI-004A effectively inhibits PGAM1 enzyme activity intumors in vivo in resected tumors from xenograft nude mice. These datatogether suggest that targeting PGAM1 by PGMI-004A inhibits PGAM1 invivo, and that this inhibition causes specific toxicity to tumor cells.

PGAM1 protein expression and enzyme activity levels are upregulated inprimary leukemia cells from diverse AML, CML and B-ALL patients (n=12),compared to control peripheral blood cells from healthy donors (n=4)(FIG. 8C). Consistent with the observations in cancer cell lines,inhibiting PGAM1 by PGMI-004A treatment results in increased 3-PG anddecreased 2-PG levels in primary leukemia cells from a representativeAML patient (FIG. 8D). PGMI-004A treatment also results in decreasedcell viability and reduced PGAM1 activity and lactate production in thesamples from 7 (1 CIVIL and 6 AML) out of 8 leukemia patients. FIG. 8Eshow results using samples from CML and AML patients as representatives,respectively. Moreover, methyl-2-PG treatment rescues the decreased cellviability (FIG. 8F; 8G left) and lactate production (FIG. 8G right) inprimary leukemia cells from representative AML patients. In addition,PGMI-004A treatment did not affect cell viability of mononucleocytes inperipheral blood samples from two healthy human donors (FIG. 8H) andCD34+ cells isolated from bone marrow samples from four healthy donors(FIG. 8I), suggesting promising anti-cancer potential of PGMI-004A withminimal toxicity to human blood cells.

PGAM1 Molecular Inhibitors

The attenuation of PGAM1 impacts tumor growth suggest PGAM1 as ananti-cancer target. A screening strategy was designed using coupledPGAM1 and enolase assays and identified three lead small moleculecompounds, including alizarin, as PGAM1 inhibitors from a library ofsmall molecule compounds obtained from the Developmental TherapeuticProgram of NIH/NCI (FIG. 9E,). Alizarin (FIG. 9E; top) is a1,2-dihydroxyanthraquinone. Treatment with alizarin results in decreasedproliferation of H1299 cells in a dose-dependent manner.

A group of alizarin derivatives were screened and found that alizarinRed S (FIG. 9E; middle) has more potent PGAM1 inhibitory potentialcompared to alizarin (FIG. 9F). Co-crystallization and structuralanalyses revealed that alizarin Red S binds to the active site of PGAM1suggesting the molecular mechanism by which alizarin and alizarin Red Sinhibit PGAM1. Modeling and analyses of unbiased Fo-Fc differencedensity maps was conducted, allowing us to determine the orientation ofalizarin Red S. Based on this analysis, the conjugated ring system ofanthracene-9,10-dione was determined to be is the main structure ofalizarin Red S, which binds to the pocket of PGAM1 catalytic site byforming hydrogen bonds between its two oxygen molecules on ring C andPGAM1 residues Phe22 and Arg116. The sulfonic acid group of alizarin RedS also forms strong hydrogen bonds with Ser23 and Arg62, which furtherstabilizes the interaction between PGAM1 and alizarin Red S.

Since both alizarin and alizarin Red S are rather hydrophilic and mayhave low cell membrane permeability, a group of alizarin Red Sderivatives were designed by adding hydrophobic groups through asulfonamide bond (FIG. 10A). Such a sulfonamide bond would be hydrolyzedby esterase inside of cells and the active sulfonic acid group would beexposed to inhibit PGAM1. These compounds were tested and identified onederivative that was named PGAM1 inhibitor 004A (PGMI-004A) (FIG. 10A;bottom). Although PGMI-004A does not show improved inhibitory effect onPGAM1 compared to alizarin and alizarin Red S in the in vitro PGAM1assay using purified recombinant PGAM1, PGMI-004A demonstrates enhancedpotency to inhibit PGAM1 in leukemia KG1a cells compared to its parentalcompounds (FIG. 10C). This may be due to the fact that PGMI-004A is morehydrophobic than alizarin and alizarin Red S which confers better cellpermeability. PGMI-004A treatment results in decreased cellproliferation of cancer cells including H1299 (FIG. 10E), KG1a (FIG.10F) and 212LN cells (FIG. 10G).

1. An antibody to PGAM1 phospho-Y26 epitope.
 2. The antibody of claim 1,wherein the antibody is a human chimera or humanized antibody.
 3. Apharmaceutical composition comprising an antibody that binds PGAM1phospho-Y26 epitope and a pharmaceutically acceptable excipient.
 4. Amethod of treatment of cancer comprising administering an effectiveamount of an antibody that binds the PGAM1 to a subject in need thereof.5. The method of claim 1 wherein the antibody binds PGAM1 residue Tyr26.6. The method of claim 1 wherein the antibody binds PGAM1phospho-tyrosine at residue Tyr26.
 7. The method of claim 4 wherein theantibody is administered in combination with another anti-cancer agent.